Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity.

The antipsychotropic drug Duloxetine rescues PAX6 haploinsufficiency of mutant limbal stem cells through inhibition of the MEK/ERK signaling pathway.

Aniridia is a panocular disease causing progressive severe visual impairment and blindness due to PAX-6 haploinsufficiency. One of the most disabling ocular symptoms is aniridia-related keratopathy (ARK), a progressive corneal opacification due to epithelial impairment, vascular and conjunctival pathologies. There is currently no available treatment to prevent progressive visual loss. For this aim, we have used mutant limbal cells for phenotypic screening using FDA-approved and bio-actives drug library and found Duloxetine, a serotonin and norepinephrine reuptake inhibitor used against severe depression as able to enhance endogenous PAX6 expression and target genes, which returned fairly to amounts found in normal limbal cells. In addition, Duloxetine could restore cell migration of the mutant cells. Furthermore, we show that Duloxetine activates PAX6 through inhibition of the ERK pathway on limbal mutant cells. This observation fits the recent report that MEK inhibitors enhance PAX6 in vivo, partially rescuing aniridia developmental phenotype of Pax6+/- mice. The discovery of an unique compound able to enhance PAX6 activity and that could be locally administered using eye drops associated with drug repurposing is expected to lead to rapid development of applicable drugs for the topical (eye drops) treatment of aniridia.

Ritanserin, a potent serotonin 2A receptor antagonist, represses MEK/ERK signalling pathway to restore PAX6 production and function in aniridia-like cellular model.

Aniridia is a panocular inherited rare eye disease linked to heterozygous mutations on the PAX6 gene, which fail to properly produce sufficient protein essential for normal eye development and function. Most of the patients suffer from aniridia-related keratopathy, a progressive opacification of the cornea. There is no effective treatment for this blinding disease. Here we screen for small compounds and identified Ritanserin, a serotonin 2A receptor antagonist, that can rescue PAX6 haploinsufficiency of mutant limbal cells, defective cell migration and PAX6-target gene expression. We further demonstrated that Ritanserin activates PAX6 production through the selective inactivation of the MEK/ERK signaling pathway. Our data strongly suggest that repurposing this therapeutic molecule could be effective in preventing or treating existing blindness by restoring corneal transparency.

Improvement of epidermal covering on AEC patients with severe skin erosions by PRIMA-1MET/APR-246.

P63 is a major transcription factor regulating skin development and homeostasis. It controls many genes involved in cell proliferation, adhesion, and early differentiation. P63 is mutated in several rare syndromes called p63-related ectodermal dysplasia syndromes (ED). The main forms are EEC and AEC syndromes due to p63 missense mutations on the DBD and SAM domains, respectively. ED patients display many developmental defects, including ectrodactyly, clef/lip palate, and ectodermal dysplasia, while AEC patients suffer from severe skin erosions that not always heal. We have previously showed that ED-derived iPSC display altered epidermal commitment. P63 belongs to the p53 gene family sharing similar structural domains. We found that ED-iPSC epidermal commitment can be rescued by a p53-reactivating compounds called PRIMA-1MET, also named APR-246 and currently used in anticancer clinical trials. Here, we established primary epidermal culture from two AEC children (S.F. and Y.M.) suffering from persistent skin erosions at age of 9 and 15, respectively. These patients carry missense mutations on the SAM domain (I576T and I537T). We found that primary keratinocytes (KCs) isolated from these AEC patients underwent altered epidermal differentiation that was rescued by PRIMA-1MET treatment. It prompted us to formulate the compound onto a cream that was topically applied on the right hand of one patient and on the scalp of the second patient. In both cases, the daily treatment allowed re-epithelialization of the eroded skin and a drastic loss of pain after few weeks, improving quality of life. Normally, mutant p63 exerts a dominant-negative effect, mainly through the formation of aggregate with WT p63 and p73. PRIMA-1MET did not reduce protein aggregation while enhancing cell differentiation, suggesting that PRIMA-1MET targets cell differentiation and not p63 activity directly. In conclusion, we propose that repurposing of the antitumoral PRIMA-1MET compound could become a general treatment of AEC skin erosions.

Modeling of Aniridia-Related Keratopathy by CRISPR/Cas9 Genome Editing of Human Limbal Epithelial Cells and Rescue by Recombinant PAX6 Protein.

Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem cell (LSC) deficiency, impaired healing response and abnormal differentiation of the corneal epithelium. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis, and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared with the parental LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Moreover, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach for aniridia-related keratopathy. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows the creation of relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches. Stem Cells 2018;36:1421-1429.

Skin absorption of six performance amines used in metalworking fluids.

Every year, 10 million workers are exposed to metalworking fluids (MWFs) that may be toxic. There are four types of MWFs: neat oils and three water-based MWFs (soluble oil, semisynthetic and synthetic), which are diluted with water and whose composition varies according to the mineral oils ratio. MWFs also contain various additives. To determine the absorption of six amines used as corrosion inhibitors and biocides in MWFs, porcine skin flow-through diffusion cell experiments were conducted with hydrophilic ethanolamines (mono-, di- and triethanolamine, MEA, DEA and TEA respectively) and a mixture of lipophilic amines (dibutylethanolamine, dicyclohexylamine and diphenylamine). The six amines were dosed in four vehicles (water and three generic water-based MWF formulations) and analyzed using a scintillation counter or gas chromatography/mass spectrometry. These 24 h studies showed that dermal absorption significantly (P < 0.05) increased from water for the six amines (e.g. 1.15 ± 0.29% dose; DEA in water) compared to other formulations (e.g. 0.13 ± 0.01% dose; DEA in semisynthetic MWF) and absorption was greatest for dibutylethanolamine in all the formulations. The soluble oil formulation tended to increase the dermal absorption of the hydrophilic amines. The permeability coefficient was significantly higher (P < 0.05) with TEA relative to the other hydrophilic amines (e.g. 4.22 × 10(-4) ± 0.53 × 10(-4) cm h(-1) [TEA in synthetic MWF] vs. 1.23 × 10(-4) ± 0.10 × 10(-4) cm h(-1) [MEA in synthetic MWF]), except for MEA in soluble oil formulation. Future research will confirm these findings in an in vivo pig model along with dermatotoxicity studies. These results should help MWF industries choose safer additives for their formulations to protect the health of metalworkers.