Head of Hearing Therapeutics Ear Science Institute Australia Perth, Western Australia, Australia
Abstract: Mutations in the USH2A gene can cause Usher syndrome type 2A, characterised by congenital hearing loss and progressive vision loss due to retinitis pigmentosa. We characterised fibroblast lines derived from patient samples containing USH2A c.949C>A and c.1256 G>T mutations, and healthy controls and generated the induced pluripotent stem cell (iPSC) lines. Fibroblasts were characterised using viability assays and immunocytochemistry; and reprogrammed to pluripotency using Oct4, Sox2, Lin28, Klf4, and L-Myc with episomal vectors. On Day 25, colonies were selected for clonal expansion and USH2A mutations confirmed using Sanger sequencing. iPSC gene expression was measured using quantitative RT-PCR; protein expression was analysed using immunocytochemistry. Fibroblast lines had typical elongated morphology, and normal cells had faster growth than patient cells. All iPSC displayed typical growth characteristics and morphologies of pluripotent stem cell colonies. Pluripotency proteins (OCT4, NANOG, SOX2 and SSEA4) and genes (OCT4, NANOG, SOX2 and KLF4) were expressed similarly in all lines. Trilineage genes (PAX6, DCX, TBXT, AFP, and SOX7) had minimal expression in undifferentiated iPSC and increased expression in embryoid bodies derived from these iPSC. Development and characterisation of iPSC lines from patients with Usher syndrome represents a unique opportunity to study differences in inner ear development from those of healthy controls.
Funding Source: Australian Government Medical Research Future Fund - Stem Cell Therapies Mission Grant, Western Australian Future Health Research and Innovation Fund, Ian Potter Foundation, Perron Charitable Foundation, Channel 7 Telethon Trust.
