A Newly Developed Chemically Defined Serum-Free Medium Suitable for Human Primary Keratinocyte Culture and Tissue Engineering Applications.

In our experience, keratinocytes cultured in feeder-free conditions and in commercially available defined and serum-free media cannot be as efficiently massively expanded as their counterparts grown in conventional bovine serum-containing medium, nor can they properly form a stratified epidermis in a skin substitute model. We thus tested a new chemically defined serum-free medium, which we developed for massive human primary keratinocyte expansion and skin substitute production. Our medium, named Surge Serum-Free Medium (Surge SFM), was developed to be used alongside a feeder layer. It supports the growth of keratinocytes freshly isolated from a skin biopsy and cryopreserved primary keratinocytes in cultured monolayers over multiple passages. We also show that keratin-19-positive epithelial stem cells are retained through serial passaging in Surge SFM cultures. Transcriptomic analyses suggest that gene expression is similar between keratinocytes cultured with either Surge SFM or the conventional serum-containing medium. Additionally, Surge SFM can be used to produce bilayered self-assembled skin substitutes histologically similar to those produced using serum-containing medium. Furthermore, these substitutes were grafted onto athymic mice and persisted for up to six months. In conclusion, our new chemically defined serum-free keratinocyte culture medium shows great promise for basic research and clinical applications.

Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells.

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, ß-catenin, GSK-3ß and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.

Influence of the Postmortem/Storage Time of Human Corneas on the Properties of Cultured Limbal Epithelial Cells.

Besides being a powerful model to study the mechanisms of corneal wound healing, tissue-engineered human corneas (hTECs) are sparking interest as suitable substitutes for grafting purposes. To ensure the histological and physiological integrity of hTECs, the primary cultures generated from human cornea (identified as human limbal epithelial cells (hLECs) that are used to produce them must be of the highest possible quality. The goal of the present study consisted in evaluating the impact of the postmortem/storage time (PM/ST) on their properties in culture. hLECs were isolated from the entire cornea comprising the limbus and central cornea. When grown as monolayers, short PM/ST hLECs displayed increased daily doublings and generated more colonies per seeded cells than long PM/ST hLECs. Moreover, hLECs with a short PM/ST exhibited a markedly faster wound closure kinetic both in scratch wound assays and hTECs. Collectively, these results suggest that short PM/ST hLECs have a greater number of highly proliferative stem cells, exhibit a faster and more efficient wound healing response in vitro, and produce hTECs of a higher quality, making them the best candidates to produce biomaterial substitutes for clinical studies.

Contribution of the STAT Family of Transcription Factors to the Expression of the Serotonin 2B (HTR2B) Receptor in Human Uveal Melanoma.

Uveal melanoma (UM) remains the most common intraocular malignancy among diseases affecting the adult eye. The primary tumor disseminates to the liver in half of patients and leads to a 6 to 12-month survival rate, making UM a particularly aggressive type of cancer. Genomic analyses have led to the development of gene-expression profiles that can efficiently predict metastatic progression. Among these genes, that encoding the serotonin receptor 2B (HTR2B) represents the most discriminant from this molecular signature, its aberrant expression being the hallmark of UM metastatic progression. Recent evidence suggests that expression of HTR2B might be regulated through the Janus kinase/Signal Transducer and Activator of Transcription proteins (JAK/STAT) intracellular signalization pathway. However, little is actually known about the molecular mechanisms involved in the abnormally elevated expression of the HTR2B gene in metastatic UM and whether activated STAT proteins participates to this mechanism. In this study, we determined the pattern of STAT family members expressed in both primary tumors and UM cell-lines, and evaluated their contribution to HTR2B gene expression. Examination of the HTR2B promoter sequence revealed the presence of a STAT putative target site (5'-TTC (N)3 GAA3') located 280 bp upstream of the mRNA start site that is completely identical to the high affinity binding site recognized by these TFs. Gene profiling on microarrays provided evidence that metastatic UM cell lines with high levels of HTR2B also express high levels of STAT proteins whereas low levels of these TFs are observed in non-metastatic UM cells with low levels of HTR2B, suggesting that STAT proteins contribute to HTR2B gene expression in UM cells. All UM cell lines tested were found to express their own pattern of STAT proteins in Western blot analyses. Furthermore, T142 and T143 UM cells responded to interleukins IL-4 and IL-6 by increasing the phosphorylation status of STAT1. Most of all, expression of HTR2B also considerably increased in response to both IL-4 and IL-6 therefore providing evidence that HTR2B gene expression is modulated by STAT proteins in UM cells. The binding of STAT proteins to the -280 HTR2B/STAT site was also demonstrated by electrophoretic mobility shift assay (EMSA) analyses and site-directed mutation of that STAT site also abolished both IL-4 and IL-6 responsiveness in in vitro transfection analyses. The results of this study therefore demonstrate that members from the STAT family of TFs positively contribute to the expression of HTR2B in uveal melanoma.

The WNK1 kinase regulates the stability of transcription factors during wound healing of human corneal epithelial cells.

Due to its superficial anatomical localization, the cornea is continuously subjected to injuries. Damages to the corneal epithelium trigger important changes in the composition of the extracellular matrix to which the basal human corneal epithelial cells (hCECs) attach. These changes are perceived by membrane-bound integrins and ultimately lead to re-epithelialization of the injured epithelium through intracellular signalin. Among the many downstream targets of the integrin-activated signaling pathways, WNK1 is the kinase whose activity is the most strongly increased during corneal wound healing. We previously demonstrated that pharmacological inhibition of WNK1 prevents proper closure of wounded human tissue-engineered cornea in vitro. In the present study, we investigated the molecular mechanisms by which WNK1 contributes to corneal wound healing. By exploiting transcription factors microarrays, electrophoretic mobility-shift assay, and gene profiling analyses, we demonstrated that the DNA binding properties and expression of numerous transcription factors (TFs), including the well-known, ubiquitous TFs specific protein 1 (Sp1) and activator protein 1 (AP1), were reduced in hCECs upon WNK1 inhibition by WNK463. This process appears to be mediated at least in part by alteration in both the ubiquitination and glycosylation status of these TFs. These changes in TFs activity and expression impacted the transcription of several genes, including that encoding the α5 integrin subunit, a well-known target of both Sp1 and AP1. Gene profiling revealed that only a moderate number of genes in hCECs had their level of expression significantly altered in response to WNK463 exposition. Interestingly, analysis of the microarray data for these deregulated genes using the ingenuity pathway analysis software predicted that hCECs would stop migrating and proliferating but differentiate more when they are grown in the presence of the WNK1 inhibitor. These results demonstrate that WNK1 plays a critical function by orienting hCECs into the appropriate biological response during the process of corneal wound healing.

Moyamoya Disease Susceptibility Gene RNF213 Regulates Endothelial Barrier Function.

BACKGROUND: Variants in the ring finger protein 213 (RNF213) gene are known to be associated with increased predisposition to cerebrovascular diseases development. Genomic studies have identified RNF213 as a major risk factor of Moyamoya disease in East Asian descendants. However, little is known about the RNF213 (ring finger protein 213) biological functions or its associated pathogenic mechanisms underlying Moyamoya disease. METHODS: To investigate RNF213 loss-of-function effect in endothelial cell, stable RNF213-deficient human cerebral endothelial cells were generated using the CRISPR-Cas9 genome editing technology. RESULTS: In vitro assays, using RNF213 knockout brain endothelial cells, showed clear morphological changes and increased blood-brain barrier permeability. Downregulation and delocalization of essential interendothelial junction proteins involved in the blood-brain barrier maintenance, such as PECAM-1 (platelet endothelial cell adhesion molecule-1), was also observed. Brain endothelial RNF213-deficient cells also showed an abnormal potential to transmigration of leukocytes and secreted high amounts of proinflammatory cytokines. CONCLUSIONS: Taken together, these results indicate that RNF213 could be a key regulator of cerebral endothelium integrity, whose disruption could be an early pathological mechanism leading to Moyamoya disease. This study also further reinforces the importance of blood-brain barrier integrity in the development of Moyamoya disease and other RNF213-associated diseases.

Contribution of the Transcription Factors Sp1/Sp3 and AP-1 to Clusterin Gene Expression during Corneal Wound Healing of Tissue-Engineered Human Corneas.

In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.

The Human Tissue-Engineered Cornea (hTEC): Recent Progress.

Each day, about 2000 U.S. workers have a job-related eye injury requiring medical treatment. Corneal diseases are the fifth cause of blindness worldwide. Most of these diseases can be cured using one form or another of corneal transplantation, which is the most successful transplantation in humans. In 2012, it was estimated that 12.7 million people were waiting for a corneal transplantation worldwide. Unfortunately, only 1 in 70 patients received a corneal graft that same year. In order to provide alternatives to the shortage of graftable corneas, considerable progress has been achieved in the development of living corneal substitutes produced by tissue engineering and designed to mimic their in vivo counterpart in terms of cell phenotype and tissue architecture. Most of these substitutes use synthetic biomaterials combined with immortalized cells, which makes them dissimilar from the native cornea. However, studies have emerged that describe the production of tridimensional (3D) tissue-engineered corneas using untransformed human corneal epithelial cells grown on a totally natural stroma synthesized by living corneal fibroblasts, that also show appropriate histology and expression of both extracellular matrix (ECM) components and integrins. This review highlights contributions from laboratories working on the production of human tissue-engineered corneas (hTECs) as future substitutes for grafting purposes. It overviews alternative models to the grafting of cadaveric corneas where cell organization is provided by the substrate, and then focuses on their 3D counterparts that are closer to the native human corneal architecture because of their tissue development and cell arrangement properties. These completely biological hTECs are therefore very promising as models that may help understand many aspects of the molecular and cellular mechanistic response of the cornea toward different types of diseases or wounds, as well as assist in the development of novel drugs that might be promising for therapeutic purposes.

Irradiated Human Fibroblasts as a Substitute Feeder Layer to Irradiated Mouse 3T3 for the Culture of Human Corneal Epithelial Cells: Impact on the Stability of the Transcription Factors Sp1 and NFI.

Because of the worldwide shortage of graftable corneas, alternatives to restore visual impairments, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. In this study, we investigated the impact of replacing irradiated 3T3 (i3T3) murine fibroblast feeder cells by irradiated human corneal fibroblasts (iHFL) on the expression of Sp1 and NFI and evaluated their contribution to the proliferative properties of human corneal epithelial cells (hCECs) in both monolayer cultures and human tissue engineered corneas (hTECs). hCECs co-cultured with iHFL could be maintained for up to two more passages than when they were grown with i3T3. Western Blot and electrophoretic mobility shift assays (EMSAs) revealed no significant difference in the feeder-layer dependent increase in Sp1 at both the protein and DNA binding level, respectively, between HCECs grown with either i3T3 or iHFL. On the other hand, a significant increase in the expression and DNA binding of NFI was observed at each subsequent passage when hCECs were co-cultured along with i3T3. These changes were found to result from an increased expression of the NFIA and NFIB isoforms in hCECs grown with i3T3. Exposure of hCECs to cycloheximide revealed an increased stability of NFIB that likely resulted from post-translational glycosylation of this protein when these cells were co-cultured with i3T3. In addition, iHFL were as efficient as i3T3 at preserving corneal, slow-cycling, epithelial stem cells in the basal epithelium of the reconstructed hTECs. Furthermore, we observed an increased expression of genes whose encoded products promote hCECs differentiation along several passages in hCECs co-cultured with either type of feeder layer. Therefore, the iHFL feeder layer appears to be the most effective at maintaining the proliferative properties of hCECs in culture most likely by preserving high levels of Sp1 and low levels of NFIB, which is known for its gene repressor and cell differentiation properties.

Grafting of an autologous tissue-engineered human corneal epithelium to a patient with limbal stem cell deficiency (LSCD).

PURPOSE: In this study, we evaluated the feasibility of recovering the corneal surface integrity in a patient suffering from unilateral LSCD through the transplantation of cultured autologous corneal epithelial cells. METHODS: Human corneal epithelial cells (HCECs) were isolated from a limbal biopsy of the contralateral eye of a patient with unilateral LSCD and cultured in monolayer in the presence of an irradiated human fibroblasts feeder layer (iHFL). To produce a cultured autologous corneal epithelium (CACE), HCECs were seeded on a fibrin substrate and maintained in culture until confluence. The in vitro obtained CACE was then used to treat the affected eye of the patient. Two years later, a successful penetrating keratoplasty was performed. RESULTS: Efficient restoration of the corneal epithelium was achieved following transplantation of CACE indicating probable re-colonization of the cornea by stem cells. Corneal transparency was restored after removing the scarred stroma by performing a penetrating keratoplasty. CONCLUSION: CACE produced in vitro was shown to restore a normal corneal surface capable of sustaining a viable and clear penetrating keratoplasty and reestablished a near normal vision in a unilateral LSCD patient.

Analysis of the proteasome activity and the turnover of the serotonin receptor 2B (HTR2B) in human uveal melanoma.

Uveal melanoma (UM), although a very rare disease, remains a particularly aggressive type of cancer as near 50% of the UM presenting patients will also develop liver metastases within 15 years from the initial diagnostic. One of the most reliable predictive markers of UM at risk of evolving toward the formation of liver lesions is an abnormally elevated level of expression of the transcript encoding the 5-Hydroxytryptamine (serotonin) receptor 2B (HTR2B). In our previous study, we demonstrated that transcription of the HTR2B gene was under the regulatory influences of two transcription factors (TFs), NFI and RUNX1. However, the action of these TFs was insufficient to explain the elevated level of the HTR2B protein in metastatic UM cells or the discrepancies we observed between its expression at the transcriptional and protein levels, therefore suggesting that additional post-translational modifications may also contribute to the altered expression of HTR2B in UM cells. In the present study, we investigated whether the turnover of HTR2B by the proteasome could account at least in part for its deregulated expression. Microarray analyses performed with UM cell lines derived from both non-metastatic and metastatic UM primary tumors revealed important alterations in the expression of some of the transcripts encoding both the E3 ubiquitin ligases and the various subunits of the proteasome, and these modifications were further exacerbated by cell passaging in culture. These alterations also correlated with significant changes in the enzymatic activity of the proteasome. However, the highest proteasome activity and amount of ubiquitinated HTR2B observed in the metastatic T142 cell line, as revealed by immunoprecipitation of ubiquitinated proteins and Western blotting using the HTR2B antibody, apparently had little impact on the total content of HTR2B protein. This contrasts with the near total disappearance of this receptor in the non-metastatic T108 cell line. Our study therefore suggests that the inability of the proteasome to degrade HTR2B in metastatic UM cells might rely on an increased stability of the ubiquitinated receptor in these cells.

Qualitatively Monitoring Binding and Expression of the Transcription Factors Sp1 and NFI as a Useful Tool to Evaluate the Quality of Primary Cultured Epithelial Stem Cells in Tissue Reconstruction.

Electrophoretic mobility shift assays and Western blots are simple, efficient, and rapid methods to study DNA-protein interactions and protein expression, respectively. Primary cultures and subcultures of epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. The preservation of stem cells through the culture process is essential to produce high quality substitutes. However, the increase in the number of cell passages is associated with a decrease in their ability to proliferate until senescence is reached. This process is likely to be mediated by the altered expression of nuclear-located transcription factors such as Sp1 and NFI, whose expression has been documented to be required for cell adhesion, migration, and differentiation. In some of our recent studies, we observed a correlation between reconstructed tissues exhibiting poor histological and structural characteristics and a low expression of Sp1 in their constituting epithelial cells. Therefore, monitoring both the expression and DNA binding of these transcription factors in human skin and corneal epithelial cells is a useful tool for characterizing the quality of primary cultured epithelial cells.