NRG1 Regulates Proliferation, Migration and Differentiation of Human Limbal Epithelial Stem Cells.

Limbal epithelial stem/progenitor cells (LESCs) proliferate, migrate and differentiate into mature corneal epithelium cells (CECs) that cover the ocular surface. LESCs play a crucial role in the maintenance and regeneration of the corneal epithelium, and their dysfunction can lead to various corneal diseases. Neuregulin 1 (NRG1) is a member of the epidermal growth factor family that regulates the growth and differentiation of epithelial tissues. Here, we depicted the dynamic transcriptomic profiles during human CEC differentiation, identifying six gene co-expression modules that were specific to different differentiation stages. We found that the expression of NRG1 was high in human LESCs and decreased dramatically upon differentiation. Knockdown of NRG1 significantly inhibited LESC proliferation and upregulated the expression of the terminal differentiation marker genes KRT3, KRT12 and CLU. In addition, the scratch wound closure assay showed that knockdown of NRG1 attenuated wound closure of LESCs over 24 h. Together, we dissected the transcriptional regulatory dynamics during CEC differentiation and identified NRG1 as a key regulator that promoted LESC proliferation and migration and maintained the undifferentiated state.

The immunology of corneal limbal stem cells: the up-to-date approach to stem cell transplantation.

Limbal epithelial stem cells (LSC, LESC) are multipotent cells used as regenerative treatment of the cornea in patients with limbal epithelial stem cell deficiency (LSCD, LESCD). There are different types of stem cell grafting including cultivated limbal epithelial transplantation (CET) and simple limbal epithelial transplantation (SLET). The outcomes of the techniques have been assessed as similar, with differences in the sample size required during the procedures. The most important culture components for stem cell cultivation include 3T3 murine fibroblasts, human amniotic membrane (HAM), fibrin gel, and culture medium. The culture medium may be enriched with serum or not; however, xenobiotic-free materials are preferred because of the low risk of pathogen transmission. Multiple studies have defined molecules important for maintaining the function of LSC including C/EBP δ, Bmi-1, p63 α, interleukins (IL-6), epithelial structural proteins - keratins, and antibodies against epidermal growth factor receptor (EGFR). The cell phenotype of LSC has been described with factors of transplantation success rate such as a high percentage of p63 positive cells. The article emphasizes the role of recipient tissue preparation, modern cultivation techniques and pathophysiological processes in LSC transplantation effectiveness.

Tissue-Restricted Stem Cells as Starting Cell Source for Efficient Generation of Pluripotent Stem Cells: An Overview.

Induced pluripotent stem cells (iPSCs) have vast biomedical potential concerning disease modeling, drug screening and discovery, cell therapy, tissue engineering, and understanding organismal development. In the year 2006, a groundbreaking study reported the generation of iPSCs from mouse embryonic fibroblasts by viral transduction of four transcription factors, namely, Oct4, Sox2, Klf4, and c-Myc. Subsequently, human iPSCs were generated by reprogramming fibroblasts as a starting cell source using two reprogramming factor cocktails [(i) OCT4, SOX2, KLF4, and c-MYC, and (ii) OCT4, SOX2, NANOG, and LIN28]. The wide range of applications of these human iPSCs in research, therapeutics, and personalized medicine has driven the scientific community to optimize and understand this reprogramming process to achieve quality iPSCs with higher efficiency and faster kinetics. One of the essential criteria to address this is by identifying an ideal cell source in which pluripotency can be induced efficiently to give rise to high-quality iPSCs. Therefore, various cell types have been studied for their ability to generate iPSCs efficiently. Cell sources that can be easily reverted to a pluripotent state are tissue-restricted stem cells present in the fetus and adult tissues. Tissue-restricted stem cells can be isolated from fetal, cord blood, bone marrow, and other adult tissues or can be obtained by differentiation of embryonic stem cells or trans-differentiation of other tissue-restricted stem cells. Since these cells are undifferentiated cells with self-renewal potential, they are much easier to reprogram due to the inherent characteristic of having an endogenous expression of few pluripotency-inducing factors. This review presents an overview of promising tissue-restricted stem cells that can be isolated from different sources, namely, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, limbal epithelial stem cells, and spermatogonial stem cells, and their reprogramming efficacy. This insight will pave the way for developing safe and efficient reprogramming strategies and generating patient-specific iPSCs from tissue-restricted stem cells derived from various fetal and adult tissues.

Peripheral (not central) corneal epithelia contribute to the closure of an annular debridement injury.

Corneal epithelia have limited self-renewal and therefore reparative capacity. They are continuously replaced by transient amplifying cells which spawn from stem cells and migrate from the periphery. Because this view has recently been challenged, our goal was to resolve the conflict by giving mice annular injuries in different locations within the corneolimbal epithelium, then spatiotemporally fate-mapping cell behavior during healing. Under these conditions, elevated proliferation was observed in the periphery but not the center, and wounds predominantly resolved by centripetally migrating limbal epithelia. After wound closure, the central corneal epithelium was completely replaced by K14+ limbal-derived clones, an observation supported by high-resolution fluorescence imaging of genetically marked cells in organ-cultured corneas and via computational modeling. These results solidify the essential role of K14+ limbal epithelial stem cells for wound healing and refute the notion that stem cells exist within the central cornea and that their progeny have the capacity to migrate centrifugally.

Effect of Conditioned Media of Limbal Epithelial Cells and Corneal Stromal Cells on Functional Activity of Limb Mesenchymal Stem Cells.

We studied the effect of conditioned media from limbal epithelial stem cells, fibroblasts, and corneal keratocytes on the functional activity of human limbal mesenchymal stem cells. It was shown that the conditioned media from limbal epithelial stem cells reduced proliferative activity and inhibited migration of limbal mesenchymal stem cells. In the conditioned media of limbal epithelial stem cells, increased concentrations of VEGF and TNFα and reduced concentration of BDNF, vimentin, and fibronectin were found. The conditioned medium from corneal stromal cells did not affect functional activity of mesenchymal stem cells in the limbus. These data contribute to the understanding of the interaction of cells in the limbal niche and with corneal cells essential for the maintenance of the cellular homeostasis in the cornea.

SPARC promotes self-renewal of limbal epithelial stem cells and ocular surface restoration through JNK and p38-MAPK signaling pathways.

The purpose of this study was to investigate the effects of secreted protein acidic and rich in cysteine (SPARC) on the maintenance of limbal epithelial stem cell (LESC) stemness and restoration of ocular surface. To determine the suitable concentration of SPARC for LESC culture, the marker expression, mitogenic effect, and holoclone-forming capacity of LESCs treated with different concentrations of SPARC were analyzed. To investigate the mechanism of SPARC's action on the preservation of LESCs stemness, the phosphorylation of related signaling pathways was evaluated by Western blotting. A corneal wound model was established to verify the function of SPARC in ocular surface repair. Consecutive subculturing, colony-forming efficiency, immunofluorescence, and 5-ethynyl-2-deoxyuridine incorporation assays indicated that 1 µg/mL SPARC was a suitable concentration to stimulate LESC proliferation and preserve their proliferative potential. Compared with a control group, 1 µg/mL SPARC effectively increased the expression of ABCG-2, Bmi-1, and Ki67, while decreasing that of CK3/12. The mitogenic effect of SPARC on LESCs was found to be mediated by the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-MAPK signaling pathways, whereas the inhibitors of JNK and p38 MAPK reduced the marker expression and mitogenic capacity of LESCs. In a corneal injury model, SPARC facilitated corneal epithelial wound healing and promoted the proliferation of p63α-positive cells both in the limbus and in the epithelial healing front. SPARC promotes proliferation while suppressing spontaneous differentiation of LESCs through JNK and p38-MAPK signaling pathways, suggesting that SPARC is a promising factor for the improvement of LESCs culture in vitro and in vivo.

Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells.

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63α, ABCG2, Ki67, Integrin ß4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) < 8 h or for 1 day with pmt > 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63α was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.

KIT ligand produced by limbal niche cells under control of SOX10 maintains limbal epithelial stem cell survival by activating the KIT/AKT signalling pathway.

Homeostasis and function of limbal epithelial stem cells (LESCs) rely on the limbal niche, which, if dysfunctional, leads to limbal epithelial stem cell deficiency (LSCD) and impaired vision. Hence, recovery of niche function is a principal therapeutic goal in LSCD, but the molecular mechanisms of limbal niche homeostasis are still largely unknown. Here, we report that the neural crest transcription factor SOX10, which is expressed in neural crest-derived limbal niche cells (LNCs), is required for LNCs to promote survival of LESCs both in vivo and in vitro. In fact, using mice with a Sox10 mutation and in vitro coculture experiments, we show that SOX10 in LNCs stimulates the production of KIT ligand (KITL), which in turn activates in LESCs the KIT-AKT signalling pathway that protects the cells against activated CASPASE 3-associated cell death. These results suggest that SOX10 and the KITL/KIT-AKT pathway play key roles in limbal niche homeostasis and LESC survival. These findings provide molecular insights into limbal niche function and may point to rational approaches for therapeutic interventions in LSCD.

Corneal epithelial biology: Lessons stemming from old to new.

The anterior surface of the eye functions as a barrier to the external environment and protects the delicate underlying tissues from injury. Central to this protection are the corneal, limbal and conjunctival epithelia. The corneal epithelium is a self-renewing stratified squamous epithelium that protects the underlying delicate structures of the eye, supports a tear film and maintains transparency so that light can be transmitted to the interior of the eye (Basu et al., 2014; Cotsarelis et al., 1989; Funderburgh et al., 2016; Lehrer et al., 1998; Pajoohesh-Ganji and Stepp, 2005; Parfitt et al., 2015; Peng et al., 2012b; Stepp and Zieske, 2005). In this review, dedicated to James Funderburgh and his contributions to visual science, in particular the limbal niche, corneal stroma and corneal stromal stem cells, we will focus on recent data on the identification of novel regulators in corneal epithelial cell biology, their roles in stem cell homeostasis, wound healing, limbal/corneal boundary maintenance and the utility of single cell RNA sequencing (scRNA-seq) in vision biology studies.

Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions.

Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.

Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction.

Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) - a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for corneal reconstruction.

Abnormal corneal epithelial maintenance in mice heterozygous for the micropinna microphthalmia mutation Mp.

We investigated the corneal morphology of adult Mp/+ mice, which are heterozygous for the micropinna microphthalmia mutation, and identified several abnormalities, which implied that corneal epithelial maintenance was abnormal. The Mp/+ corneal epithelium was thin, loosely packed and contained goblet cells in older mice. Evidence also suggested that the barrier function was compromised. However, there was no major effect on corneal epithelial cell turnover and mosaic patterns of radial stripes indicated that radial cell movement was normal. Limbal blood vessels formed an abnormally wide limbal vasculature ring, K19-positive cells were distributed more widely than normal and K12 was weakly expressed in the peripheral cornea. This raises the possibilities that the limbal-corneal boundary was poorly defined or the limbus was wider than normal. BrdU label-retaining cell numbers and quantitative clonal analysis suggested that limbal epithelial stem cell numbers were not depleted and might be higher than normal. However, as corneal epithelial homeostasis was abnormal, it is possible that Mp/+ stem cell function was impaired. It has been shown recently that the Mp mutation involves a chromosome 18 inversion that disrupts the Fbn2 and Isoc1 genes and produces an abnormal, truncated fibrillin-2(MP) protein. This abnormal protein accumulates in the endoplasmic reticulum (ER) of cells that normally express Fbn2 and causes ER stress. It was also shown that Fbn2 is expressed in the corneal stroma but not the corneal epithelium, suggesting that the presence of truncated fibrillin-2(MP) protein in the corneal stroma disrupts corneal epithelial homeostasis in Mp/+ mice.

Electrospun poly(l-lactide-co-dl-lactide) nanofibrous scaffold as substrate for ex vivo limbal epithelial cell cultivation.

Ultrathin electrospun poly (l-lactide-co-dl-lactide) nanofibrous membranes coated with fibronectin were explored as scaffolds for the ex vivo cultivation of limbal epithelial cells (LECs) for the treatment of limbal stem cell deficiency. The developed scaffolds were compared with the "gold-standard" fibrin gel. The resulting membranes composed of nanofibers possessed a very low thickness of 4 µm and allowed very good optical transparency in the wet state. The fibronectin-coated nanofibrous scaffolds demonstrated LEC expansion and successful cultivation similar to that on fibrin gel. Unlike the regular cobblestone epithelial cell morphology on the fibrin gel, the nanofibrous scaffold presented a mostly irregular epithelial morphology with a shift to a mesenchymal phenotype, as confirmed by the upregulation of profibroblastic genes: ACTA2 (p = 0.023), FBLN1 (p < 0.001), and THY1 (p < 0.001). Both culture conditions revealed comparable expression of stem cell markers, including KLF4, ΔNp63α and ABCG2, emphasizing the promise of polylactide-based nanofibrous membranes for further investigations.

The Potential Reversible Transition between Stem Cells and Transient-Amplifying Cells: The Limbal Epithelial Stem Cell Perspective.

Stem cells (SCs) undergo asymmetric division, producing transit-amplifying cells (TACs) with increased proliferative potential that move into tissues and ultimately differentiate into a specialized cell type. Thus, TACs represent an intermediary state between stem cells and differentiated cells. In the cornea, a population of stem cells resides in the limbal region, named the limbal epithelial stem cells (LESCs). As LESCs proliferate, they generate TACs that move centripetally into the cornea and differentiate into corneal epithelial cells. Upon limbal injury, research suggests a population of progenitor-like cells that exists within the cornea can move centrifugally into the limbus, where they dedifferentiate into LESCs. Herein, we summarize recent advances made in understanding the mechanism that governs the differentiation of LESCs into TACs, and thereafter, into corneal epithelial cells. We also outline the evidence in support of the existence of progenitor-like cells in the cornea and whether TACs could represent a population of cells with progenitor-like capabilities within the cornea. Furthermore, to gain further insights into the dynamics of TACs in the cornea, we outline the most recent findings in other organ systems that support the hypothesis that TACs can dedifferentiate into SCs.

The Role of Insulin-like Growth Factor (IGF) System in the Corneal Epithelium Homeostasis-From Limbal Epithelial Stem Cells to Therapeutic Applications.

The corneal epithelium, comprising three layers of cells, represents the outermost portion of the eye and functions as a vital protective barrier while concurrently serving as a critical refractive structure. Maintaining its homeostasis involves a complex regenerative process facilitated by the functions of the lacrimal gland, tear film, and corneal nerves. Crucially, limbal epithelial stem cells located in the limbus (transitional zone between the cornea and the conjunctiva) are instrumental for the corneal epithelium integrity by replenishing and renewing cells. Re-epithelialization failure results in persistent defects, often associated with various ocular conditions including diabetic keratopathy. The insulin-like growth factor (IGF) system is a sophisticated network of insulin and other proteins essential for numerous physiological processes. This review examines its role in maintaining the corneal epithelium homeostasis, with a special focus on the interplay with corneal limbal stem cells and the potential therapeutic applications of the system components.

Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells.

Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

ABCB5+ Limbal Epithelial Stem Cells Inhibit Developmental but Promote Inflammatory (Lymph) Angiogenesis While Preventing Corneal Inflammation.

The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.

Limbal Epithelial Stem Cells in the Diabetic Cornea.

Continuous replenishment of the corneal epithelium is pivotal for maintaining optical transparency and achieving optimal visual perception. This dynamic process is driven by limbal epithelial stem cells (LESCs) located at the junction between the cornea and conjunctiva, which is otherwise known as the limbus. In patients afflicted with diabetes, hyperglycemia-induced impairments in corneal epithelial regeneration results in persistent epithelial and other defects on the ocular surface, termed diabetic keratopathy (DK), which progressively diminish vision and quality of life. Reports of delayed corneal wound healing and the reduced expression of putative stem cell markers in diabetic relative to healthy eyes suggest that the pathogenesis of DK may be associated with the abnormal activity of LESCs. However, the precise role of these cells in diabetic corneal disease is poorly understood and yet to be comprehensively explored. Herein, we review existing literature highlighting aberrant LESC activity in diabetes, focusing on factors that influence their form and function, and emerging therapies to correct these defects. The consequences of malfunctioning or depleted LESC stocks in DK and limbal stem cell deficiency (LSCD) are also discussed. These insights could be exploited to identify novel targets for improving the management of ocular surface complications that manifest in patients with diabetes.

Wnt/ß-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo.

Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/ß-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (TP63), progenitor (SOX9), quiescence (CEBPD), and proliferation (MKI67, PCNA) genes and the upregulation of genes for differentiation (CX43, KRT3) in treated- compared to non-treated samples. The pathway activation was shown by AXIN2 upregulation and enhanced levels of accumulated ß-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/ß-catenin signaling profile demonstrated an upregulation of WNT1, WNT3, WNT5A, WNT6, and WNT11 gene expression and a downregulation for WNT7A and DKK1 in the treated samples. No significant differences were found for WNT2, WNT16B, WIF1, and DKK2 gene expression. Overall, our results demonstrate that activation of Wnt/ß-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.

Recent Approaches to the Modification of Collagen Biomatrix as a Corneal Biomatrix and Its Cellular Interaction.

Over the last several decades, numerous modifications and advancements have been made to design the optimal corneal biomatrix for corneal epithelial cell (CECs) or limbal epithelial stem cell (LESC) carriers. However, researchers have yet to discover the ideal optimization strategies for corneal biomatrix design and its effects on cultured CECs or LESCs. This review discusses and summarizes recent optimization strategies for developing an ideal collagen biomatrix and its interactions with CECs and LESCs. Using PRISMA guidelines, articles published from June 2012 to June 2022 were systematically searched using Web of Science (WoS), Scopus, PubMed, Wiley, and EBSCOhost databases. The literature search identified 444 potential relevant published articles, with 29 relevant articles selected based on inclusion and exclusion criteria following screening and appraising processes. Physicochemical and biocompatibility (in vitro and in vivo) characterization methods are highlighted, which are inconsistent throughout various studies. Despite the variability in the methodology approach, it is postulated that the modification of the collagen biomatrix improves its mechanical and biocompatibility properties toward CECs and LESCs. All findings are discussed in this review, which provides a general view of recent trends in this field.