Induced pluripotent stem cell (iPSC) line from an epidermolysis bullosa simplex patient heterozygous for keratin 5 E475G mutation and with the Dowling Meara phenotype

Nikola Kolundzic,Preeti Khurana,Carl Hobbs,Marija Rogar,Sandra Ropret,Hans Törmä,Dusko Ilic,Mirjana Liovic

doi:10.1016/j.scr.2019.101424

Abstract

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex.

Highlights

We have generated MLi002-A, a new induced pluripotent stem cell line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation
A library of human induced pluripotent stem cell (iPSC) lines with KRT 5 and 14 gene mutations can be used in basic studies of disease mechanism and protein function and to engineer highly specific in vitro 3D skin models (Petrova et al, 2014) for drug discovery purposes of new potential therapies for EBS
In order to verify that the K5 p.E475G (c.1424A > G) mutation was retained, the iPSCs were screened with restriction enzyme digestion followed by Sanger sequencing (Fig. 1C)

Summary

Introduction

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. MLi002-A iEBS Faculty of Medicine, University of Ljubljana, Slovenia Mirjana Liovic, mirjana.liovic@mf.uni-lj.si iPSC Human Age: not known Sex: Female Ethnicity: Caucasian Epidermal keratinocytes Mixed Non-integrating SeV-mediated delivery of OCT4, SOX2, c-MYC and KLF4 NO N/A Epidermolysis bullosa simplex DowlingMeara OMIM #131760 Keratin 5 gene (KRT5), locus 12q13.13, autosomal dominant loss-of-function mutation NM_000424.3(KRT5):c.1424A > G (K5 p.E475G) N/A N/A

Results

Conclusion