Generation And Characterization Of Deafness Patient-specific Induced Pluripotent Stem Cells With Mitochondrial DNA A1555G Mutation

Hearing loss is a common congenital disorder. Nowadays, there are360million patients with deafness around the world. There are more than30million patients in china. Hearing loss can be caused by genetic and environmental factors. About50%of the deafness cases have a genetic predisposition. mtDNA12S rRNA A1555G mutation is one of the main causes in aminoglycoside-induced non-syndromic hearing loss. Since mitochondrial diseases owes a characteristic of multiple copy and double membrane, it lacks a practical technique to particularly modify mtDNA, while constructing relevant animal model is in a tough period. All of these seriously restrict the research of pathogenesis of mitochondrial disease.Induced pluripotent stem cells (iPS cells or iPSCs) are a type of pluripotent stem cells artificially derived from adult somatic cells with four transcription factors transgene. Patient-specific iPS cells are designed to induce patient’s somatic cells into iPS cells. Since it is originated from individuals affected different disease, it surmounts species difference existing in animal models. Additionally, the source of primary cells is extensive, which overcomes the difficulty in sampling. Moreover, patient-specific iPS cells maintain a patient’s genome especially the defect gene. All the advantages of patient-specific iPS cells provide disease researches with a perfect cell model.In this paper we collected and cultured in vitro the exploited renal tubular epithelial cells from urine of the hearing loss patient with mtDNA A1555G mutation and a normal control who has the same mitochondrial genome haplotype, establishing patient’s urine cell lines harboring mtDNA A1555G mutation and the normal urine cell lines. Then we delivered four transcriptional factors (Oct-4, Sox-2, c-Myc, Klf4) into urine cells, generating deafness-specific iPS cells and normal iPS cells. Through microscope examination, the generated iPS cell lines exhibited compact clones which are similar to ES cells including a round shape, large nucleoli, and scant cytoplasm. Alkaline phosphatase, Real-time PCR, bisulfite genomic sequencing analyses immunofluorescence, semi-quantitative PCR and G-banding chromosome analysis indicated that these generated iPS cells were all positive for alkaline phosphatase; expressed higher endogenous ESC genes compared with urine cells while negligible transgenes; were highly unmethylated while in urine cells they were highly methylated; expressed hES cell-specific surface antigens, including SSEA-3, SSEA-4, tumor-related antigen TRA-1-60, TRA-1-81and NANOG protein; showed integration of the exogenous transgenes in the genome; had a normal karyotype. in vivo induction of embryoid body (EB) and in vitro formation of teretoma demonstrated the iPS clones had the developmental potential to give rise to differentiated derivatives of all three primary germ layers. Meanwhile, mitochondrial genome sequencing of deafness-specific iPS cells revealed that iPS cells still maintained the mtDNA A1555G mutation and no other new mutations arised compared to parental urine cells. Transmissinon electron microscope (TEM) showed the mitochondria were unmatured with indistinguishable cristae in both deafness-specific and normal iPS cells.Above all, this research sccusfully established deafness-specific iPS cell lines with mtDNA A1555G mutation. It can provide a reliable cell model to deeply clarify this disease’s pathogenesis as well as drug screening, and open up a new road for diagnose or treatment for hearing loss.