Background: Mutations in USH2A can cause Usher syndrome type 2A, characterised by congenital hearing loss and progressive vision loss due to retinitis pigmentosa.
Aims: Characterise fibroblast lines derived from patient samples containing USH2A c.949C>A and c.1256 G>T mutations, and healthy controls. Next, generate and characterise iPSC lines.
Methods: 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 qRT-PCR; protein expression was analysed using immunocytochemistry.
Results: 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 SSEA-4) 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.
Conclusion: 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.