Essential Role of CD63 in Maintaining Corneal Epithelial Identity in the Human Limbus
Yuzuru Sasamoto, Kosei Suzuki, Shinri Sato, Catherine A. A. Lee, Gabrielle Martin, Bruce R. Ksander, Markus H. Frank, Natasha Y. Frank
TL;DR
This study shows that CD63 is crucial for maintaining corneal epithelial identity and function in the human limbus.
Contribution
The study identifies CD63 as a novel functional component of limbal stem cell biology and corneal epithelial homeostasis.
Findings
CD63 is highly expressed in the human limbus and is required for maintaining corneal epithelial cell identity.
CD63 knockdown reduces cell proliferation and expression of corneal epithelium-specific genes, including PAX6.
CD63 co-expresses with ABCB5 and is linked to limbal stem cell markers in murine corneal epithelial cells.
Abstract
Building on the identification of ABCB5 as a marker of limbal stem cells (LSCs), this study examines CD63, a newly identified molecule co-expressed with ABCB5 in limbal epithelial cells, to define its role in maintaining corneal epithelial cell identity. RNA sequencing (RNA-seq) was performed on flow cytometry–sorted Abcb5-positive and Abcb5-negative murine corneal epithelial cells. CD63 expression in human corneal tissue was assessed by immunostaining. CD63 was silenced in cultured human limbal epithelial cells using siRNA-mediated knockdown and resulting molecular and cellular changes were analyzed by qRT-PCR, flow cytometry, RNA-seq, Western blotting, and cell proliferation assays. RNA-seq analysis revealed increased expression of LSC markers, including Krt15, Krt6b, Fgfr1, Gpha2, Ifitm3, Ifitm1, and Cd63, and decreased expression of differentiation-associated markers, such as…
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Taxonomy
TopicsCorneal Surgery and Treatments · Proteoglycans and glycosaminoglycans research · Developmental Biology and Gene Regulation
The cornea forms the transparent, outermost barrier of the eye and plays a crucial role in maintaining normal vision. Structurally, it is composed of five anatomically distinct layers: the epithelium, Bowman's membrane, stroma, Descemet's membrane, and endothelium, arranged sequentially from the surface inward. Each layer contributes uniquely to the cornea's transparency, strength, and function. The corneal epithelium, derived from surface ectoderm, is characterized by a unique gene expression profile that distinguishes it from conjunctival and cutaneous epithelia.1 Central to this identity is PAX6, a master regulator of ocular development, which orchestrates the expression of key corneal epithelial genes, including keratin 12 (KRT12), keratin 3 (KRT3), clusterin (CLU), aldehyde dehydrogenase 3A1 (ALDH3A1), angiopoietin-like 7 (ANGPTL7), and transketolase (TKT).2^,^3 In addition, a cornea-associated crystallin, ALDH1A1, transforming growth factor β-induced (TGFBI), implicated in corneal dystrophies, and the recently discovered myeloma overexpressed gene (MYEOV) are also abundantly expressed in the corneal epithelium.4^–^7 Collectively, these molecular features confer the corneal epithelium its distinct functional and structural characteristics.
The limbus, located at the junction between the cornea and conjunctiva, serves as the niche for limbal stem cells (LSCs), which are essential for the lifelong maintenance and regeneration of the corneal epithelium.8^,^9 Through our previous work, we identified ABCB5 as a marker of LSCs in both mouse and human corneas.9^,^10 More recently, we demonstrated that ABCB5-positive LSCs give rise to BCAM-positive transit amplifying cells (TACs), which migrate centripetally and superficially to repopulate the corneal surface and generate the fully differentiated epithelial layers.11^,^12
In this study, we performed RNA sequencing (RNA-seq) of the flow cytometry-sorted Abcb5-positive LSCs and Abcb5-negative limbal cells to define the molecular signature of Abcb5-positive LSCs in mice. Among the identified markers, we prioritized CD63, a molecule that has not been previously studied in the human cornea. We demonstrated that CD63 is highly expressed in the human limbus and is essential for maintaining corneal epithelium-specific proteins, at least in part by regulating PAX6.
Materials and Methods
Rodent Cell Source
Eyes were harvested from 3-month-old male C57BL/6 mice in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. To isolate the corneal epithelium, whole globes were incubated overnight at 4°C in Dispase I (FUJIFILM Wako Chemicals, Osaka, Japan). The epithelial layer was then dissociated into single cells by treatment with TrypLE Express Enzyme (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 30 minutes.
Human Cell Source
Human whole eye globes and corneas were obtained from the Saving Sight (Kansas City, MO, USA) and CorneaGen (Seattle, WA, USA) eye banks. The human tissue experiments were conducted in accordance with the ARVO Best Practices guidelines for the use of human eye tissue in research (November 2021). Donor characteristics are described in Supplementary Table S1. Whole globes were fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA, USA) at 4°C overnight, then transferred to 70% ethanol until paraffin embedding at the Brigham and Women’s Hospital Pathology Core. Single-cell suspensions of limbal epithelium were prepared from sclerocorneal tissues as previously described.11^,^13 Briefly, the central cornea was excised using an 8 mm disposable biopsy punch (Integra LifeSciences, Plainsboro, NJ, USA), and the corneal endothelium was mechanically removed. The remaining limbal tissue was incubated in PluriSTEM Dispase II Solution (MilliporeSigma, Burlington, MA, USA) at 37°C for 1 hour. Epithelial cells were then scraped from the tissue and further dissociated with TrypLE Express Enzyme (Thermo Fisher Scientific) at 37°C for 30 minutes. The resulting single cells were cultured in DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 10 ng/mL keratinocyte growth factor (KGF; PeproTech, Rocky Hill, NJ, USA), 10 µM Y-27632 (Tocris Bioscience, Bristol, UK), and B-27 Supplement (Thermo Fisher Scientific).14
Flow Cytometry
Dissociated mouse corneal epithelial cells were resuspended in phosphate-buffered saline (PBS; GE Healthcare Life Sciences, Marlborough, MA, USA) supplemented with 2% fetal bovine serum (FBS; Thermo Fisher Scientific). Cells were incubated with 40 µg/mL human anti-Abcb5 monoclonal antibody (clone 3B9)10 at 37°C for 30 minutes, followed by incubation with Donkey Anti-Human IgG H+L (DyLight 650; Thermo Fisher Scientific) on ice for 30 minutes. To deplete hematopoietic cells, the suspension was stained with 0.1 µg/mL PE-conjugated anti-CD45 monoclonal antibody (Abcam, Cambridge, UK) and 3 µg/mL PE-conjugated anti-CD11b monoclonal antibody (clone REA592; Miltenyi Biotec, Bergisch Gladbach, Germany) on ice for 30 minutes. Dead cells were excluded by staining with 30 nM SYTOX Green Dead Cell Stain (Thermo Fisher Scientific) on ice for 20 minutes.
Cultured human limbal epithelial cells were stained with 5 µg/mL VioBlue-conjugated anti-CD63 monoclonal antibody (Miltenyi Biotec). Viability was assessed using GloCell Fixable Viability Dye Violet 780 (Stemcell Technologies, Vancouver, Canada) at 4°C for 30 minutes. Cell sorting was performed using a FACSAria II cell sorter (BD Biosciences, San Jose, CA, USA), and flow cytometric analysis was conducted on a FACSCelesta flow cytometer (BD Biosciences). Data were analyzed using BD FACSDiva Software version 9.0 and FlowJo version 10.5.0 (BD Biosciences).
Colony-Forming Assay
The colony-forming efficiency was evaluated according to a previously reported protocol.11^,^13 Briefly, 500 trypsinized cells were seeded onto mitomycin C-treated 3T3-J2 feeder layers in 6-well plates. Cells were cultured for 10 days in keratinocyte culture medium (KCM) supplemented with 10 ng/mL KGF and 10 µM Y-27632. After 10 days, colonies were fixed in 10% neutral-buffered formalin and visualized with rhodamine B staining.
Cell Proliferation Assay
Cell proliferation was assessed using the Click-iT Plus EdU Flow Cytometry Kit (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. Briefly, cells were incubated with 20 µM 5-ethynyl-2′-deoxyuridine (EdU) for 12 hours prior to harvesting. EdU incorporation was subsequently quantified using a FACSymphony A3 flow cytometer (BD Biosciences).
RNA Sequencing
Total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany) in combination with the DNA-free DNA Removal Kit (Thermo Fisher Scientific). For mouse Abcb5-positive and -negative corneal epithelial cells, mRNA libraries were prepared using the SMART-Seq version 4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories, Mountain View, CA, USA). Sequencing was performed on the Illumina NextSeq 500 platform (single-end, 75 bp) at the Molecular Biology Core Facility (MBCF) of Dana-Farber Cancer Institute, and RNA-seq counts were generated using Salmon.15
For CD63 knockdown human limbal epithelial cells, RNA samples were submitted to Zymo Research (Irvine, CA, USA) for RNA-seq. Following rRNA depletion,16 mRNA libraries were constructed using the Zymo-Seq RiboFree Total RNA Library Prep Kit (Zymo Research) and sequenced on the Illumina NovaSeq platform (150 bp paired-end). Sequence reads were aligned using the STAR aligner (version 2.6.1d),17 and gene expression quantification was performed with EdgeR.18 Differential gene expression (DEG) and Gene Ontology (GO) term enrichment analyses were conducted using DESeq2.19
Immunofluorescence Staining
Paraffin-embedded tissues were sectioned at 5-µm thickness using a microtome. After deparaffinization and antigen retrieval, permeabilization and blocking were performed by Tris-buffered saline (TBS; Boston BioProducts, Ashland, MA, USA) containing 5% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and 0.3% Triton X-100 (MilliporeSigma) at room temperature for 30 minutes. The sections were then incubated with mouse anti-KRT12 mAb (1:100, sc-515882; Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-KRT13 pAb (1:200, PA5-19049; Thermo Fisher Scientific), and rabbit anti-CD63 mAb (1:300, ab252919; Abcam) at 4°C overnight. After washing with TBS twice, the sections were incubated with Alexa Fluor 488-conjugated mouse secondary antibody (1:400; Abcam), Alexa Fluor 568-conjugated rabbit secondary antibody (1:400; Abcam), and Alexa Fluor 647-conjugated goat secondary antibody (1:400; Abcam) at room temperature for 1 hour and with Hoechst 33342 (Thermo Fisher Scientific) at room temperature for 10 minutes. After the wash with TBS, the sections were sealed with ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan) was used to take images, which were analyzed by NIS-Elements AR version 5.30.06 (Nikon).
RNA Interference
RNA interference was performed by using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) as previously described.20^,^21 Transfected Silencer Select siRNAs (Thermo Fisher Scientific) were: Silencer Select Negative Control No. 1 siRNA (bioinformatically designed, non-targeting siRNA), CD63 siRNAs (s2699 and s2700), and PAX6 siRNAs (s529238, s529239, s529237, s10067, and s10068).
Reverse Transcription and Quantitative PCR
The cDNA synthesis was performed by the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). The qPCR was performed with TaqMan Gene Expression Assay probes (Thermo Fisher Scientific) and TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific). GAPDH (Hs99999905_m1), CD63 (Hs01041237_g1), PAX6 (Hs01088114_m1), KRT12 (Hs00165015_m1), CLU (Hs00971656_m1), ALDH1A1 (Hs00946916_m1), ALDH3A1 (Hs00964880_m1), and TGFBI (Hs00932747_m1) TaqMan probes were used. The cycling conditions were 95°C for 20 seconds and 50 cycles of [95°C/1 second; 60°C/20 seconds]. Relative gene expression was calculated using GAPDH as a reference gene. The miRNA detection was performed using the TaqMan MicroRNA Assay (miR-184 [#000485] and U6 [#001973]; Thermo Fisher Scientific) and TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). The PCR cycling conditions were 50°C for 2 minutes, 95°C for 20 seconds, and 50 cycles of [95°C/3 seconds; 60°C/30 seconds]. Relative gene expression was calculated using U6 as a reference gene.
Western Blot Analyses
The siRNA-treated limbal epithelial cells were harvested using RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with cOmplete Protease Inhibitor Cocktail (MilliporeSigma), and the lysates were incubated on ice for 30 minutes. After the debris was removed by centrifugation, protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). The lysates were mixed with SDS-sample buffer (Boston BioProducts) and 2-mercaptoethanol (MilliporeSigma) and were denatured at 95°C for 10 minutes. After SDS-PAGE gel electrophoresis, the separated proteins were transferred on the PVDF blotting membranes (GE Healthcare Life Sciences). Membrane blocking was performed by TBS with Tween 20 (MilliporeSigma) containing 5% blotting-grade blocker (Bio-Rad) at room temperature for 1 hour, and incubated with primary antibodies at 4°C overnight. Primary antibodies used in the current study were: rabbit anti-β-actin pAb (1:1000; Cell Signaling Technology; 4970), rabbit anti-CLU mAb (1:1000; Abcam; ab92548), mouse anti-ALDH1A1 mAb (1:10000; Thermo Fisher Scientific; MA5-34924), goat anti-ALDH3A1 pAb (1:25000; GeneTex; GTX88085), goat anti-TGFBI pAb (1:1000; GeneTex; GTX88213), rabbit anti-KRT12 mAb (1:5000; Abcam; ab185627), and rabbit anti-PAX6 mAb (1:1000; Abcam; ab195045). After the wash with TBS-T, the membranes were incubated with HRP-conjugated mouse (Cell Signaling Technology), rabbit (Cell Signaling Technology), or goat (R&D Systems, Minneapolis, MN, USA) secondary antibodies at room temperature for 1 hour. Protein signal development was performed by using Western Lightning Plus-ECL (PerkinElmer, Waltham, MA, USA) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). ChemiDoc MP Imaging System (Bio-Rad) was used to take images, and Image Lab software version 5.2.1 (Bio-Rad) was used to measure the expression levels. The protein expression levels were normalized to that of β-actin.
miRNA Transfection
The miRNA transfection was performed by transfecting mirVana miRNA mimic (Thermo Fisher Scientific) by using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) at the same time as transfecting siRNAs. The miRNAs used were: mirVana miRNA Mimic, Negative Control #1, and hsa-miR-184 (#MC10207).
Statistical Analysis
The data are presented as mean ± standard deviation (SD). Dunnett’s test was performed to compare the siRNA-treated samples with the negative control samples (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
Data Availability
The RNA-seq data discussed in this publication were deposited in the Gene Expression Omnibus under accession number GSE308189.
Results
Cd63 is Highly Expressed in Mouse Abcb5-Positive LSCs
The entire mouse corneal epithelium was isolated, and flow cytometry was used to purify Abcb5-positive LSCs and Abcb5-negative corneal cells (Figs. 1A, 1B, Supplementary Fig. S1A). RNA-seq of these sorted populations identified 827 genes significantly upregulated, and 691 genes downregulated in Abcb5-positive LSCs compared with Abcb5-negative cells (P < 0.001; Fig. 1C, Supplementary Tables S2, S3). GO enrichment analysis demonstrated that Abcb5-positive LSCs showed increased expression of genes associated with stemness, including “response to wounding” and “cell-cell adhesion via plasma-membrane adhesion molecule.” In contrast, genes related to proliferation and migration were predominantly upregulated in Abcb5-negative limbal epithelial cells (Supplementary Figs. S1B, S1C). Abcb5-positive LSCs also exhibited higher expression of established LSC markers such as Krt15, Krt6b, and Fgfr1, and lower expression of corneal epithelial markers, including Krt12 and Gja1 (Fig. 1D). Additionally, Abcb5-positive LSCs expressed elevated levels of Gpha2, Ifitm3, and Cd63, which have been identified as additional markers of quiescent LSCs.22 A recent study by Jiang et al. identified high Ifitm1 expression in LSCs and early transient amplifying (eTA) cells and described its role in promoting cell proliferation through the inhibition of Ovol1.23 Consistent with these findings, our RNA-seq analysis revealed upregulation of Ifitm1 and downregulation of Ovol1 in Abcb5-positive cells (see Fig. 1D).
*Cd63 is highly expressed in mouse Abcb5-positive limbal stem cells (LSCs). (A) Schematic illustration of Abcb5-positive LSC and Abcb5-negative corneal cell isolation from mouse eyes. (B) Representative Abcb5-positive LSC and Abcb5-negative corneal cell isolation by flow cytometry (n = 3). FSC, forward scatter; SSC, side scatter; A, area; H, height; W, width; mAb, monoclonal antibody. (C) A volcano plot depicting differentially expressed genes (DEGs) in Abcb5-positive LSCs identified by RNA-seq. (D) Bar graphs illustrate expression of LSC-positive markers (Krt15, Krt6b, Fgfr1, Gpha2, Ifitm3, Cd63, and Ifitm1) and LSC-negative markers (Krt12, Gja1, and Ovol1) identified by RNA-seq (n = 3, mean ± SD, **adjusted P < 0.01, ***adjusted P < 0.0001).
CD63 is Strongly Expressed in the Human Limbal Epithelium and Regulates Key Genes Essential for Corneal Epithelial Identity
Among the genes highly expressed in Abcb5-positive LSCs, CD63 was selected for further investigation due to its previously uncharacterized function in the human limbal epithelium. Immunostaining demonstrated that CD63 is predominantly localized to the basal layers of the limbal epithelium, with significantly lower expression observed in the basal layers of the conjunctiva and in the central cornea (Fig. 2A). To clarify the functional role of CD63, siRNA-mediated knockdown (KD) was performed in cultured human limbal epithelial cells using two independent siRNAs (CD63 KD#1 and CD63 KD#2). Efficient reduction of CD63 was confirmed at both the RNA and protein levels (see Figs. 2B, 2C). Although the two KD conditions exhibited different potencies, CD63 depletion consistently inhibited cell proliferation, as evidenced by decreased colony formation (Fig. 2D) and reduced EdU uptake (Fig. 2E). RNA-seq analysis of CD63 KD cells identified 73 genes consistently downregulated and 12 genes upregulated across both KD conditions (Fig. 2F, Supplementary Tables S4, S5). Several downregulated genes are established markers of corneal epithelial identity, including KRT12, CLU, ALDH1A1, ALDH3A1, TGFBI, and MYEOV (Fig. 2G). Importantly, CD63 KD did not induce mesenchymal marker expression, suggesting that CD63 is not involved in epithelial-mesenchymal transition (Supplementary Fig. S2). Western blot analysis further confirmed the downregulation of KRT12, CLU, ALDH1A1, ALDH3A1, and TGFBI at the protein level following CD63 KD (Fig. 2H).
*CD63 is expressed in the human limbal epithelium and regulates corneal epithelial gene expression. (A) Representative KRT12 (corneal epithelial cell marker, green), KRT13 (conjunctival epithelial marker, yellow), and CD63 (red) immunostaining in the human conjunctiva, limbus, and central cornea. Hoechst 33342 (blue) was used for the nuclei staining (n = 3 donors). Scale bar = 50 µm. (B) The bar graph represents CD63 RNA expression in CD63 siRNA-treated cultured limbal epithelial cells (n = 4 donors, mean ± SD; ***P < 0.001, ****P < 0.0001). (C, left panel) Representative flow cytometry analyses of CD63 protein expression in CD63 siRNA-treated cultured limbal epithelial cells detected by flow cytometry. (C, right panel) The bar graph represents quantitative analyses of CD63 protein expression determined by flow cytometry (n = 3 donors, mean ± SD; *P < 0.05). (D, left panel) Representative macroscopic images of colonies formed by CD63 KD cells compared to the control siRNA-transfected cells. (D, right panel) The bar graph represents a comparative analysis of the colony-forming efficiency (n = 6 donors, mean ± SD; *P < 0.05, **P < 0.01). (E) Comparative analyses of the percentage of EdU-positive proliferating cells by CD63 KD in cultured human limbal epithelial cells (n = 5 donors, mean ± SD; **P < 0.01). (F) A Venn diagram illustrates the overlap of differentially expressed genes identified in both CD63 KD#1 and CD63 KD#2 conditions. (G) Bar graphs represent normalized counts of CD63, KRT12, CLU, ALDH1A1, ALDH3A1, TGFBI, and MYEOV RNA expression in CD63 siRNA-treated cultured limbal epithelial cells (n = 3 donors, mean ± SD, *adjusted P < 0.05, **adjusted P < 0.01, ***adjusted P < 0.001, ****adjusted P < 0.0001). (H, left panel) Western blot analyses of KRT12, CLU, ALDH1A1, ALDH3A1, and TGFBI expression in control and CD63 siRNA-treated limbal epithelial cells. (H, right panel) Bar graphs represent quantitative analyses of protein expression (n = 4 donors, mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.01, ***P < 0.0001).
CD63 Maintains Corneal Epithelial Phenotype by Regulating PAX6 Expression
Although RNA-seq did not reveal a significant reduction in PAX6 transcript levels following CD63 KD (Supplementary Fig. S3A), Western blot analysis showed that PAX6 protein expression was reduced by half in CD63 KD cells (CD63 KD#1: 53.1 ± 24.0%, P = 0.0485; CD63 KD#2: 44.8 ± 17.3%, P = 0.0128, n = 4 donors; Fig. 3A). Furthermore, PAX6 KD in cultured limbal epithelial cells led to decreased expression of KRT12, CLU, ALDH1A1, and ALDH3A1 at both RNA and protein levels (Fig. 3B, Supplementary Fig. S3B). These findings suggest that the effects of CD63 KD on these corneal epithelial genes are at least partially mediated through PAX6.
*CD63 regulates corneal epithelial phenotype by maintaining PAX6 expression. (A, left panel) Western blot analysis of PAX6 expression in control and CD63 siRNA-treated limbal epithelial cells. (A, right panel) The bar graph represents quantitative analyses of PAX6 protein expression (n = 4 donors, mean ± SD; *P < 0.05). (B, left panel) Western blot analysis of PAX6, KRT12, CLU, ALDH1A1, ALDH3A1, and TGFBI expressions in control and PAX6 siRNA-treated limbal epithelial cells. (B, right panel) Bar graph represents quantitative analyses of protein expression (n = 3 donors, mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (C) Bar graph represents miR-184 expression by CD63 siRNA-treated cultured limbal epithelial cells (n = 3 donors, mean ± SD; **P < 0.01, **P < 0.001). (D) Bar graphs represent normalized counts of FOG2 and NUS1 RNA expression in CD63 siRNA-treated cultured limbal epithelial cells (n = 3 donors, mean ± SD).
Because KD of miR-184 is known to reduce PAX6 expression,24 miR-184 levels were assessed by quantitative RT-PCR and found to be significantly downregulated in CD63 KD cells (CD63 KD#1: 10.2 ± 4.1%, P = 0.0011; CD63 KD#2: 8.7 ± 1.4%, P = 0.0001, n = 3 donors; Fig. 3C). RNA-seq analysis confirmed upregulation of its target genes, FOG2 and NUS1 (Fig. 3D). However, overexpression of miR-184 failed to restore PAX6 levels in CD63 KD limbal epithelial cells (see Supplementary Figs. S3C, S3D), suggesting that the reduction of PAX6 following CD63 KD is not solely mediated by miR-184 downregulation.
Discussion
This study identified that mouse corneal Abcb5-positive LSCs express the recently discovered quiescent LSC markers, such as Gpha2 and Cd63,22^,^23 supporting their quiescent stem cell identity observed in vivo.10 In humans, CD63 was found to be predominantly expressed in the basal epithelial cells of the limbus*.* In vitro functional assays demonstrated that CD63 positively regulates cell proliferation and plays a role in the maintenance of corneal epithelial characteristics through the regulation of the master transcription factor PAX6. These findings highlight CD63 as a key mediator of corneal epithelial homeostasis and a potential therapeutic target for limbal stem cell deficiency.
Abcb5-positive cells isolated from the mouse cornea exhibited robust expression of established LSC markers, including Krt15,25 Krt6b, and Fgfr1,26 while displaying low levels of differentiated corneal epithelial markers such as Krt12 and Gja1.27^,^28 Recent work by Altshuler et al. identified Gpha2, Ifitm3, and Cd63 as specific markers of the outer quiescent LSC population in the mouse cornea.22 Consistent with these findings, purified Abcb5-positive LSCs demonstrated high expression of these quiescent LSC markers, further supporting previous observations that ABCB5 marks quiescent LSC populations in both human and mouse cornea.10 Additionally, Abcb5-positive cells expressed high levels of Ifitm1 and low levels of Ovol1. This expression pattern was recently identified as a mechanism for the expansion of the stem/eTA cell population after corneal wounding.23
Although Cd63 expression in the limbus has been previously demonstrated in mouse models,22 its expression pattern in the human ocular surface remains insufficiently characterized. The present study demonstrates that CD63 is predominantly present in basal epithelial cells in the human limbus. KD of CD63 in cultured human limbal epithelial cells resulted in reduced cell proliferation, indicating that CD63 is involved in maintaining the high proliferative potential of LSCs upon activation. Furthermore, CD63 KD reduced the expression of several corneal epithelium-enriched genes, including KRT12, CLU, ALDH1A1, ALDH3A1, TGFBI, and MYEOV. Notably, PAX6 expression was also decreased by approximately half relative to normal levels. This observation is consistent with a previous report in Xenopus, where mutant Cd63 microinjection led to reduced Pax6 expression in the eye.29 Given that PAX6 haploinsufficiency is known to cause a spectrum of ocular defects, including progressive limbal stem cell deficiency,30^–^33 it is likely that the downregulation of corneal epithelial genes following CD63 KD is at least partially attributable to reduced PAX6 levels. Supporting this, we confirmed that PAX6 KD in cultured limbal epithelial cells led to decreased expression of KRT12, CLU, ALDH1A1, and ALDH3A1. These findings are consistent with previous studies showing that KRT12 and CLU are upregulated by PAX6 overexpression,2 and that ALDH1A1 and ALDH3A1 are downregulated in heterozygous mutant PAX6 cells.34^,^35 Although TGFBI has been reported to be upregulated in heterozygous mutant PAX6 cells,36 our results showed no significant change in TGFBI expression following PAX6 KD, suggesting that CD63 may regulate TGFBI through a PAX6-independent mechanism.
Although miR-184, a known positive regulator of PAX6,24 was also downregulated following CD63 KD, restoration of miR-184 levels was insufficient to rescue PAX6 expression. This suggests that miR-184 is not the sole pathway through which CD63 regulates PAX6 in limbal epithelial cells. Notably, miR-184 has also been shown to inhibit corneal vascularization by targeting pro-angiogenic factors, such as FOG2 and NUS1.37 Therefore, the presence of CD63-positive cells in the limbus may contribute to the maintenance of corneal avascularity, at least in part, by sustaining miR-184 expression and thereby limiting vascular invasion toward the central cornea.
CD63 is a member of the tetraspanin family and is widely recognized as a marker of extracellular vesicles (EVs).38^,^39 In the cornea, CD63-positive EVs secreted by epithelial cells function as paracrine messengers that can traverse the basement membrane to influence the underlying stroma, where they induce myofibroblast transformation from fibroblasts and keratocytes.40^,^41 CD63 interacts with TIMP-1 to activate the PI3K pathway, independent of Akt phosphorylation, in melanoma cells and the ERK signaling pathway in breast epithelial cells, thereby inhibiting apoptosis.42^,^43 Additionally, elevated TIMP-1 levels influence neutrophil homeostasis via CD63-mediated signaling.44 However, the specific signaling downstream of CD63 in the limbal epithelium remains incompletely elucidated. Moreover, no distinct corneal phenotype has been reported in relation to CD63 mutations to date. This may be due to compensation by the other tetraspanins, such as CD81 and CD9.38^,^45
In summary, beyond its role in promoting limbal epithelial cell proliferation, CD63 is critical for maintaining the limbal and corneal epithelial phenotype, at least in part through regulation of PAX6. CD63 may also influence corneal epithelial gene expression via its effects on membrane vesicle dynamics. Further studies are warranted to elucidate how CD63-mediated vesicular pathways regulate corneal epithelial-specific genes.
Supplementary Material
Supplement 1
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