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

Abstract

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. STEM CELLS

Rat limbal niche cells can induce transdifferentiation of oral mucosal epithelial cells into corneal epithelial-like cells in vitro

“…Very recently, keratoplasty lenticules, a novel carrier, have been proven to be suitable for transplantation to reconstruct the ocular surface and treat LSCD [ 42 ]. Our research team has succeeded in culturing OMECs on acellular porcine corneal stroma (APCS) [ 24 ], and a clinical trial in our hospital has demonstrated that APCS transplantation is useful for treating fungal keratitis [ 43 ].…”

Analysis and pharmacological modulation of senescence in human epithelial stem cells

“…All EEC mutants showed defects in stratification and differentiation and shorter lifespans in vitro. 20,21 Nevertheless, in this study we demonstrate that H-LESCs potency and length of lifespan are at least three times lower than H-SESCs (Figure 2), a likely explanation of the early signs of degeneration seen in the ocular surface of patients with EEC syndrome.…”

“…This is likely due to the epithelium of the skin being constantly subjected to stress and continuously renewed. 52 In our previous studies, [19][20][21] we provided evidence that a major factor in the pathophysiology of EEC syndrome is the premature degeneration of the limbal epithelial SCs and the palisade structures.…”

“…If SCs are exhausted too quickly, or if genetic defects alter their proliferative potential, tissue atrophy and premature ageing can arise. For example, mutations in the p63 gene, known to cause ectrodactyly‐ectodermal dysplasia‐clefting (EEC) syndrome, induce a rapid exhaustion of clonogenic and self‐renewal potential of ESCs, resulting in accelerated ageing 19–21 . Conversely, mutations that promote frequent SC divisions without an appropriate differentiation balance can result in abnormal tissue development and even cancer 22 …”

“…For example, mutations in the p63 gene, known to cause ectrodactylyectodermal dysplasia-clefting (EEC) syndrome, induce a rapid exhaustion of clonogenic and self-renewal potential of ESCs, resulting in accelerated ageing. [19][20][21] Conversely, mutations that promote frequent SC divisions without an appropriate differentiation balance can result in abnormal tissue development and even cancer. 22 In this study, we demonstrate that the frequency and timing of SC divisions are tightly regulated and specific for each tissue, as well as preserved in vitro to ensure a defined lifelong maintenance of the SCs population.…”