Optimized Protocol for Regeneration of the Conjunctival Epithelium Using the Cell Suspension Technique.

PURPOSE: To develop autologous tissue-engineered conjunctival epithelial sheets to be used as advanced therapy medicinal products for severe ocular surface disorders involving the conjunctiva. METHODS: Methods used aimed at 1) mapping the conjunctiva for identification of the stem cell location, 2) establishing proper cell culturing conditions, 3) identifying the proper scaffold, and 4) characterizing the conjunctival grafts better. For these purposes, immunostaining and PAS staining, serial cultivation of cells, and quantitative polymerase chain reaction ([INCREMENT]Np63α and MUC5AC) were performed. RESULTS: The inferior fornix represents the ideal area where to take the conjunctival biopsies from, with at least +3.58% of clonogenic colonies and higher percentages of stem cells compared with other areas, as confirmed by [INCREMENT]Np63α expression levels (6.79% ± 1.18%). The standard culture conditions are necessary when cells are cultured on bare plastic, while animal-free media can be used for conjunctival cell culture on the scaffold. Fibrin glue represents the ideal scaffold for production of epithelial conjunctival grafts because it allows physiological expression of the main conjunctival cell markers, with K19 as the ideal one (98.5% ± 0.5% positive cells). The presence of goblet cells (6.3% ± 1.3%) and expression of the stem cell marker [INCREMENT]Np63α (1.65% ± 0.35% positive cells) were also assessed. CONCLUSIONS: Our findings pave the way for ex vivo cultivation of conjunctival epithelial cells onto a scaffold using the cell suspension technique by means of animal-free media. This would allow us to obtain conjunctival grafts for clinical purposes, thus giving a therapeutic option to patients with conjunctival diseases refractory to current therapies.

Oral Mucosa-Derived Induced Pluripotent Stem Cells from Patients with Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare monogenic disease with autosomal dominant inheritance caused by mutations in the TP63 gene, leading to progressive corneal keratinocyte loss, limbal stem cell deficiency (LSCD), and eventually blindness. Currently, there is no treatment available to cure or slow down the keratinocyte loss. Human oral mucosal epithelial stem cells (hOMESCs), which are a mixed population of keratinocyte precursor stem cells, are used as source of autologous tissue for treatment of bilateral LSCD. However, hOMESCs from EEC patients have a reduced life span due to TP63 mutations and cannot be used for autologous transplantation. Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of autologous limbal stem cell for EEC patients and can be genetically modified by genome editing technologies to correct the disease ex vivo before transplantation. In this study, we describe for the first time the generation of integration-free EEC-hiPSCs from hOMESCs of EEC patients by Sendai virus vector and episomal vector-based reprogramming. The generated hiPSC clones expressed pluripotency markers and were successfully differentiated into derivatives of the three germ layers, as well as toward corneal epithelium. These cells may be used for EEC disease modeling and open perspectives for applications in cell therapy of LSCD.

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction.

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum "XerumFree™ XF205" (XF); (3) CnT-20 medium supplemented with "XerumFree™ XF205" (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ∆Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ∆Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ∆Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.

Cytology of the healthy canine and feline ocular surface: comparison between cytobrush and impression technique.

BACKGROUND: Impression cytology (IC) is a noninvasive technique in which filters are used to sample superficial layers of ocular epithelium. OBJECTIVES: The aim of this study was to compare cytology specimens obtained by IC and cytobrush from healthy canine and feline eyes. METHODS: Dogs and cats were prospectively sampled using polytetrafluorethylene filters on the right eye, and cytobrush on the left eye. Wright-Giemsa-stained specimens were evaluated by 2 observers. Cellularity, preservation, and morphology of cells and presence of goblet and inflammatory cells were scored with a 4-grade scale. Inter-observer agreement and effects of topical anesthesia were analyzed. RESULTS: In 20 canine IC samples, 10 showed good cellularity (score 2-3) and 13 good preservation. Superficial epithelial cells (SEC) were present in 13/20 of IC, while basal-intermediate cells (BIC) were seen in 14/20. In 6/20 and 7/20, goblet and inflammatory cells were noted, respectively. In 20 cats, 15 of IC showed good cellularity and 14 good preservation, and SEC were present in 16/20 of IC and BIC in 17/20. In 13/20 and 3/20 cats, goblet cells and inflammatory cells were noted, respectively. Canine cytobrush specimens appeared well preserved (9/20) and had good cellularity (8/20). In feline cytobrush specimens, good preservation and cellularity were observed in 16/20 and 14/20, respectively. In both species, all cell types were present without a clear separation. There was moderate to fair agreement about cellular morphology in IC between observers. Specimens obtained with and without anesthesia were comparable. CONCLUSION: Impression cytology allowed collection of samples with maintained cytoarchitecture, while cytoplasmatic and nuclear details were often difficult to evaluate.

Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

UNLABELLED: : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. SIGNIFICANCE: This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome.

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600.