Generation of 3D lacrimal gland organoids from human pluripotent stem cells.

Lacrimal glands are the main exocrine glands of the eyes. Situated within the orbit, behind the upper eyelid and towards the temporal side of each eye, they secrete lacrimal fluid as a major component of the tear film. Here we identify cells with characteristics of lacrimal gland primordia that emerge in two-dimensional eye-like organoids cultured from human pluripotent stem cells1. When isolated by cell sorting and grown under defined conditions, the cells form a three-dimensional lacrimal-gland-like tissue organoid with ducts and acini, enabled by budding and branching. Clonal colony analyses indicate that the organoids originate from multipotent ocular surface epithelial stem cells. The organoids exhibit notable similarities to native lacrimal glands on the basis of their morphology, immunolabelling characteristics and gene expression patterns, and undergo functional maturation when transplanted adjacent to the eyes of recipient rats, developing lumina and producing tear-film proteins.

Co-ordinated ocular development from human iPS cells and recovery of corneal function.

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

Generation and validation of a PITX2-EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells.

PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator-like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2-IRES2-EGFP sequence downstream of the stop codon in exon 8 of PITX2 Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2-EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.

Diverse Localization Patterns of an R-Type Lectin in Marine Annelids.

Lectins facilitate cell-cell contact and are critical in many cellular processes. Studying lectins may help us understand the mechanisms underlying tissue regeneration. We investigated the localization of an R-type lectin in a marine annelid (Perinereis sp.) with remarkable tissue regeneration abilities. Perinereis nuntia lectin (PnL), a galactose-binding lectin with repeating Gln-X-Trp motifs, is derived from the ricin B-chain. An antiserum was raised against PnL to specifically detect a 32-kDa lectin in the crude extracts from homogenized lugworms. The antiserum detected PnL in the epidermis, setae, oblique muscle, acicula, nerve cord, and nephridium of the annelid. Some of these tissues and organs also produced Galactose (Gal) or N-acetylgalactosamine (GalNAc), which was detected by fluorescent-labeled plant lectin. These results indicated that the PnL was produced in the tissues originating from the endoderm, mesoderm, and ectoderm. Besides, the localizing pattern of PnL partially merged with the binding pattern of a fluorescent-labeled mushroom lectin that binds to Gal and GalNAc. It suggested that PnL co-localized with galactose-containing glycans in Annelid tissue; this might be the reason PnL needed to be extracted with haptenic sugar, such as d-galactose, in the buffer. Furthermore, we found that a fluorescein isothiocyanate-labeled Gal/GalNAc-binding mushroom lectin binding pattern in the annelid tissue overlapped with the localizing pattern of PnL. These findings suggest that lectin functions by interacting with Gal-containing glycoconjugates in the tissues.

Localization and osteoblastic differentiation potential of neural crest-derived cells in oral tissues of adult mice.

In embryos, neural crest cells emerge from the dorsal region of the fusing neural tube and migrate throughout tissues to differentiate into various types of cells including osteoblasts. In adults, subsets of neural crest-derived cells (NCDCs) reside as stem cells and are considered to be useful cell sources for regenerative medicine strategies. Numerous studies have suggested that stem cells with a neural crest origin persist into adulthood, especially those within the mammalian craniofacial compartment. However, their distribution as well as capacity to differentiate into osteoblasts in adults is not fully understood. To analyze the precise distribution and characteristics of NCDCs in adult oral tissues, we utilized an established line of double transgenic (P0-Cre/CAG-CAT-EGFP) mice in which NCDCs express green fluorescent protein (GFP) throughout their life. GFP-positive cells were scattered like islands throughout tissues of the palate, gingiva, tongue, and buccal mucosa in adult mice, with those isolated from the latter shown to form spheres, typical cell clusters composed of stem cells, under low-adherent conditions. Furthermore, GFP-positive cells had markedly increased alkaline phosphatase (a marker enzyme of osteoblast differentiation) activity and mineralization as shown by alizarin red staining, in the presence of bone morphogenetic protein (BMP)-2. These results suggest that NCDCs reside in various adult oral tissues and possess potential to differentiate into osteoblastic cells. NCDCs in adults may be a useful cell source for bone regeneration strategies.

Identification of BST2 as a conjunctival epithelial stem/progenitor cell marker.

The conjunctival epithelium consists of conjunctival epithelial cells and goblet cells derived from conjunctival epithelial stem/progenitor cells. However, the source of these cells is not well known because no specific markers for conjunctival epithelial stem/progenitor cells have been discovered. Therefore, to identify conjunctival epithelial stem/progenitor cell markers, we performed single-cell RNA sequencing of a conjunctival epithelial cell population derived from human-induced pluripotent stem cells (hiPSCs). The following conjunctival epithelial markers were identified: BST2, SLC2A3, AGR2, TMEM54, OLR1, and TRIM29. Notably, BST2 was strongly positive in the basal conjunctival epithelium, which is thought to be rich in stem/progenitor cells. Moreover, BST2 was able to sort conjunctival epithelial stem/progenitor cells from hiPSC-derived ocular surface epithelial cell populations. BST2-positive cells were highly proliferative and capable of successfully generating conjunctival epithelial sheets containing goblet cells. In conclusion, BST2 has been identified as a specific marker of conjunctival epithelial stem/progenitor cells.

Ocular instillation of conditioned medium from mesenchymal stem cells is effective for dry eye syndrome by improving corneal barrier function.

Dry eye syndrome (DES) is a chronic ocular disease that induces epithelial damage to the cornea by decreasing tear production and quality. Adequate treatment options have not been established for severe DES such as Sjogren's syndrome due to complicated pathological conditions. To solve this problem, we focused on the conditioned medium of human adipose-derived mesenchymal stem cells (hAdMSC-CM), which have multiple therapeutic properties. Here, we showed that hAdMSC-CM suppressed Benzalkonium Chloride (BAC)-induced cytotoxicity and inflammation in human corneal epithelial cells (hCECs). In addition, hAdMSC-CM increased the expression level and regulated the localisation of barrier function-related components, and improved the BAC-induced barrier dysfunction in hCECs. RNA-seq analysis and pharmacological inhibition experiments revealed that the effects of hAdMSC-CM were associated with the TGFß and JAK-STAT signalling pathways. Moreover, in DES model rats with exorbital and intraorbital lacrimal gland excision, ocular instillation of hAdMSC-CM suppressed corneal epithelial damage by improving barrier dysfunction of the cornea. Thus, we demonstrated that hAdMSC-CM has multiple therapeutic properties associated with TGFß and JAK-STAT signalling pathways, and ocular instillation of hAdMSC-CM may serve as an innovative therapeutic agent for DES by improving corneal barrier function.

Neural crest-derived multipotent cells in the adult mouse iris stroma.

The purpose of this study was to characterize neural crest-derived cells within the adult murine iris. The iris was isolated from P0-Cre/Floxed-EGFP transgenic (TG) mice. The isolated iris cells formed EGFP-positive spheres on non-adhesive culture plates. Immunostaining showed that these EGFP-positive spheres expressed neural crest markers including Sox10 and p75NTR, and these cells showing in vitro sphere-forming ability were originally resided in the iris stroma (IS), in vivo. Real-time RT-PCR showed that the EGFP-positive spheres expressed significantly higher levels of the neural crest markers than EGFP-negative spheres and bone marrow-derived mesenchymal stem cells. Furthermore, the iris stromal sphere had capability to differentiate into various cell lineages including smooth muscle and cartilage. These data indicate that neural crest-derived multipotent cells can be isolated from the murine IS and expanded in sphere culture.

PAX6-positive microglia evolve locally in hiPSC-derived ocular organoids.

Microglia are the resident immune cells of the central nervous system (CNS). They govern the immunogenicity of the retina, which is considered to be part of the CNS; however, it is not known how microglia develop in the eye. Here, we studied human-induced pluripotent stem cells (hiPSCs) that had been expanded into a self-formed ectodermal autonomous multi-zone (SEAM) of cells that partially mimics human eye development. Our results indicated that microglia-like cells, which have characteristics of yolk-sac-like linage cells, naturally develop in 2D eye-like SEAM organoids, which lack any vascular components. These cells are unique in that they are paired box protein 6 (PAX6)-positive, yet they possess some characteristics of mesoderm. Collectively, the data support the notion of the existence of an isolated, locally developing immune system in the eye, which is independent of the body's vasculature and general immune system.

Long-term survival in non-human primates of stem cell-derived, MHC-unmatched corneal epithelial cell sheets.

When corneal epithelial stem cells residing in the corneal limbus become dysfunctional, called a limbal stem cell deficiency (LSCD), corneal transparency is decreased, causing severe vision loss. Transplantation of corneal epithelial cell sheets (CEPS) derived from stem cells, including induced pluripotent stem cells, is a promising treatment for LSCD. However, the potential effect of human leukocyte antigen (HLA) concordance on CEPS transplantation has not been addressed. Here, we show that there is no difference in the immune response to CEPS between HLA-matched and -unmatched peripheral blood mononuclear cells in mixed lymphocyte reactions. CEPS transplantation in cynomolgus monkeys revealed that the immune response to major histocompatibility-unmatched CEPS was not strong and could be controlled by local steroid administration. Furthermore, programmed death ligand 1 was identified as an immunosuppressive molecule in CEPS under inflammatory conditions in vitro. Our results indicate that corneal epithelium has low immunogenicity and allogeneic CEPS transplantation requires mild immunosuppression.

Induction of putative stratified epithelial progenitor cells in vitro from mouse-induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells generally exhibit a normal karyotype, are transcriptionally and epigenetically similar to embryonic stem (ES) cells, and maintain the potential to differentiate into derivatives of all germ layers. Recently, the use of different types of cell or tissue derived from iPS cells for transplantation has become a possibility. However, the differentiation of epithelial lineages from iPS cells has not yet been demonstrated. We attempted to establish a culture technique for the induction of epithelial progenitors from mouse iPS cells. Mouse iPS cells were cultured on dishes coated with type IV collagen in keratinocyte culture medium (KCM) supplemented with or without bone morphogenic protein-4 (BMP-4) or combined with pretreatment of retinoic acid (RA) and BMP-4 in the undifferentiated state. Markers for undifferentiated cells (Oct3/4, Nanog) and for differentiation (p63, cytokeratin14) were analyzed by immunofluorescence staining and real-time RT-PCR. Putative epithelial progenitors were successfully induced in vitro from iPS cells. These progenitors expressed p63, a transcription factor necessary for maintenance of regenerative epithelia and cytokeratin 14 constitutively present in the basal layer of stratified epithelia. Enhancement of putative epithelial progenitor commitment was observed when cultured in KCM with BMP-4 following pretreatment of RA and BMP-4. The differentiation efficiency of putative epithelial progenitors from iPS cell cultures was similar to that of ES cell cultures. This report is the first to demonstrate in vitro differentiation of iPS cells into putative epithelial progenitors. These iPS-derived putative epithelial progenitors provide a powerful tool for understanding the mechanisms of epithelial lineage differentiation.

[Generation of Multiple Ocular Lineages from Human Pluripotent Stem Cells and Its Application to Regenerative Medicine].

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and corneal stroma have a neural crest origin. Recent work with pluripotent stem cells (PSCs) in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. We recently demonstrated the generation from human induced pluripotent stem cells (iPSCs) of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. The concentric SEAM mimics whole-eye development because cell location within different zones is indicative of ocular cell lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. Therefore, SEAM represents a promising resource for new research of ocular morphogenesis and development. Moreover, we successfully isolated corneal epithelial progenitor cells and fabricated corneal epithelial tissue from PSCs. This approach has translational potential for treating severe corneal epithelial disease by transplantation of PSC-derived corneal epithelial tissue. To evaluate the efficacy and safety of the corneal epithelial tissue, we have started a first-in-human clinical study for patients with corneal epithelial stem cell deficiency, which began last year.

Generation of functional conjunctival epithelium, including goblet cells, from human iPSCs.

The conjunctival epithelium, which covers the sclera (the white of the eye) and lines the inside of the eyelids, is essential for mucin secretion and the establishment of a healthy tear film. Here, we describe human conjunctival development in a self-formed ectodermal autonomous multi-zone (SEAM) of cells that were derived from human-induced pluripotent stem cells (hiPSCs) and mimic whole-eye development. Our data indicate that epidermal growth factor (EGF) drives the generation of cells with a conjunctival epithelial lineage. We also show that individual conjunctival cells can be sorted and reconstituted by cultivation into a functional conjunctival epithelium that includes mucin-producing goblet cells. Keratinocyte growth factor (KGF), moreover, is necessary for the maturation of hiPSC-derived conjunctival epithelium-particularly the goblet cells-indicating key complementary roles of EGF and KGF in directing the differentiation and maturation, respectively, of the human conjunctival epithelium.

Human iPS cells engender corneal epithelial stem cells with holoclone-forming capabilities.

Human induced pluripotent stem cells (hiPSCs) can generate a multiplicity of organoids, yet no compelling evidence currently exists as to whether or not these contain tissue-specific, holoclone-forming stem cells. Here, we show that a subpopulation of cells in a hiPSC-derived corneal epithelial cell sheet is positive for ABCB5 (ATP-binding cassette, sub-family B, member 5), a functional marker of adult corneal epithelial stem cells. These cells possess remarkable holoclone-forming capabilities, which can be suppressed by an antibody-mediated ABCB5 blockade. The cell sheets are generated from ABCB5+ hiPSCs that first emerge in 2D eye-like organoids around six weeks of differentiation and display corneal epithelial immunostaining characteristics and gene expression patterns, including sustained expression of ABCB5. The findings highlight the translational potential of ABCB5-enriched, hiPSC-derived corneal epithelial cell sheets to recover vision in stem cell-deficient human eyes and represent the first report of holoclone-forming stem cells being directly identified in an hiPSC-derived organoid.

Ocular surface ectoderm instigated by WNT inhibition and BMP4.

We sought to elucidate how and when the ocular surface ectoderm commits to its differentiation into the corneal epithelium in eye development from human induced pluripotent stem cells (hiPSCs) under the influence of WNT signaling and the actions of BMP4. These signals are key drivers ocular surface ectodermal cell fate determination. It was discovered that secreted frizzled related protein-2 (SFRP2) and Dickkopf1 (DKK1), which are expressed in neural ectoderm, are both influential in the differentiation of hiPSCs, where they act as canonical WNT antagonists. BMP4, moreover, was found to simultaneously initiate non-neural ectodermal differentiation into a corneal epithelial lineage. Combined treatment of hiPSCs with exogenous BMP4 aligned to WNT inhibition for the initial four days of differentiation increased the ocular surface ectodermal cell population and induced a corneal epithelial phenotype. Specification of a surface ectodermal lineage and its fate is thus determined by a fine balance of BMP4 exposure and WNT inhibition in the very earliest stages of human eye development.

Cell-Type-Specific Adhesiveness and Proliferation Propensity on Laminin Isoforms Enable Purification of iPSC-Derived Corneal Epithelium.

A treatment for intractable diseases is expected to be the replacement of damaged tissues with products from human induced pluripotent stem cells (hiPSCs). Target cell purification is a critical step for realizing hiPSC-based therapy. Here, we found that hiPSC-derived ocular cell types exhibited unique adhesion specificities and growth characteristics on distinct E8 fragments of laminin isoforms (LNE8s): hiPSC-derived corneal epithelial cells (iCECs) and other non-CECs rapidly adhered preferentially to LN332/411/511E8 and LN211E8, respectively, through differential expression of laminin-binding integrins. Furthermore, LN332E8 promoted epithelial cell proliferation but not that of the other eye-related cells, leading to non-CEC elimination by cell competition. Combining these features with magnetic sorting, highly pure iCEC sheets were fabricated. Thus, we established a simple method for isolating iCECs from various hiPSC-derived cells without using fluorescence-activated cell sorting. This study will facilitate efficient manufacture of iCEC sheets for corneal disease treatment and provide insights into target cell-specific scaffold selection.

Generation of knockout rabbits with X-linked severe combined immunodeficiency (X-SCID) using CRISPR/Cas9.

Severe immunodeficient mice are widely used to examine human and animal cells behaviour in vivo. However, mice are short-lived and small in size; while large animals require specific large-scale equipment. Rabbits are also commonly employed as experimental models and are larger than mice or rats, easy to handle, and suitable for long-term observational and pre-clinical studies. Herein, we sought to develop and maintain stable strains of rabbits with X-linked severe combined immunodeficiency (X-SCID) via the CRISPR/Cas9 system targeting Il2rg. Consequently, X-SCID rabbits presented immunodeficient phenotypes including the loss of T and B cells and hypoplasia of the thymus. Further, these rabbits exhibited a higher success rate with engraftments upon allogeneic transplantation of skin tissue than did wild type controls. X-SCID rabbits could be stably maintained for a minimum of four generations. These results indicate that X-SCID rabbits are effective animals for use in a non-rodent model of severe immunodeficiency.

Chondroitin Sulfate as a Potential Modulator of the Stem Cell Niche in Cornea.

Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells.

Effects of nonsteroidal anti-inflammatory drugs on the expression and function of P-glycoprotein/MDR1 in Caco-2 cells.

The aim of this study was to examine the effects of 16 kinds of nonsteroidal anti-inflammatory drugs (NSAIDs) on P-glycoprotein/MDR1 in Caco-2 cells as an intestinal epithelial cell model. Cells were treated with NSAIDs for 24 hours, and then, the expression of MDR1 mRNA was evaluated by reverse-transcriptase polymerase chain reaction. The function of MDR1 in cells pretreated with NSAIDs for 48 hours was evaluated by measuring the cellular amount of rhodamine123, which is a substrate of MDR1. The expression of MDR1 mRNA was increased by diclofenac, fenbufen, indomethacin, and nimesulide and the tended to be increased by meloxicam, mepirizole, and sulindac. However, pretreatment for 48 hours with diclofenac, indomethacin, or nimesulide, but not fenbufen, resulted in a significant increase in the amount of rhodamine123 accumulated. Although NSAIDs without effects on the expression of MDR1 mRNA altered the accumulation of rhodamine123 significantly, the efflux of rhodamine123 from cells was unchanged. In conclusion, the expression of MDR1 mRNA in Caco-2 cells was demonstrated to be increased by treatment with some NSAIDs, although the transport function of MDR1 was unchanged. These findings imply that the NSAIDs did not cause the drug interaction via MDR1 induction.

KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells via endogenous TGF-ß2 suppression.

INTRODUCTION: Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-ß signalling pathway, which is involved in corneal epithelial wound healing. METHODS: HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-ß secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-ß2 treatment. RESULTS: KLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-ß canonical signalling pathway, TGF-ß2 secretion by HCECs increased up to 5 folds, and several TGF-ß-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-ß signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-ß2 treatment. CONCLUSIONS: These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-ß2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-ß signalling.