The Generation, Gene Correction And Hair Cell Differentiation Of The IPS Cells Derived From The Deafness Patient With Mutations In MYO7A

Hearing loss is a common disease in clinic. A majority of deafness people are suffering from the sensorineural deafness, which results from the irreversible damage of inner ear hair cells, auditory nerve and auditory centers at all levels. For treating sensorineural deafness, hearing-aid and artificial cochlea implant may be effective methods. However, these methods can’t solve the problem completely. There are only 150,000 hair cells in the hunman cochlea and this number would decrease because of the over stimulation of the noisy environment, chemical therapy, the use of aminoglycoside and aging. Hair cells in the organ of Corti are located in the closed environment of cochlea, and the loss of mammalian inner ear hair cells is unable to regenerate, which makes it troublesome for us to study the molecular mechanisms of hair cell development.The induced pluripotent stem cells (iPSCs) technology offers an unique opportunity to solve this problem and makes it possible to cure diseases that previously incurable such as deafness and hearing loss. iPSCs have the same pluripotency as embryonic stem cells and can be generated from several kinds of somatic cells. In theory, once given certain differentiation condition, iPSCs could be induced to differentiate into almost all kinds of cells in vitro. The iPSCs technology avoids the ethical issues brought by the studies with the use of human embryonic stem cells, rules out the possibility of immunological rejection during transplantation and makes the personalized medicine possible. What’s more, the patient-derived iPSCs could become the ideal model for the study of pathogenesis and pharmaceutical screening. Until now, somatic cells from patients suffering from several kinds of desease have been induced into iPSCs, but none of them was from deafness patient. Therefore, in this study, we generated the iPSCs from the deafness patient with compound heterozygous mutations in MYO7A and completed the gene correction and the hair cell differentiation of this iPSCs.Part one:Firstly, urine cells were separated from the urine of the normal, deafness patient with compound heterozygous mutations in MYO7A and the patient’s father. Secondly, iPSCs were established from these three kinds of urine cells. The induced iPSCs had the same morphology with human embryonic cells and were positive for alkaline phosphatase staining. They could express the pluripotent genes and proteins, form embryoid body in vitro expressing specific genes of three germ layers and form teratoma in vivo with three germ layer tissues. Lastly, karyotype analysis proved that the reprogramming didn’t influence the chromosome structure. All these results demonstrated that the urine cells derived from the deafness patient could also be induced into iPSCs with pluripotent potentials.Part two:In the cochlea, MYO7A is only expressed in hair cells. In order to study the function of this gene in hair cells and the deafness mechanism, we induced the normal derived (C-iPS)、patient derived (P-iPS) and patient’s father derived (CF-iPS) iPSCs to differentiate into otic progenitors and then into the inner ear hair cell-like cells. After culture in otic progenitor differentiation conditions for 12 days, all the three iPSC lines could differentiate into two kinds of otic progenitors respectively:the otic epithelial progenitors (OEPs) and otic neural progenitors (ONPs). The detection of early otic specific genes and proteins proved that all the three kinds of iPSCs could differentiate into otic progenitors without significant difference. Then, the otic progenitors derived from three kinds of iPSCs were proceeded to hair cell differentiation through culture in hair cell differentiation conditions with EGF and retinoic acid. After 3 weeks of differentiation, all the hair cell-like cells derived from C-iPSCs% CF-iPSCs and P-iPSCs had no significant difference in hair cell-specific genes expression and proteins immuno-labeling. But the P-iPSC-derived hair cell-like cells had much lower rate in FM1-43 endocytosis and the stereociliary protruding from the cell surface couldn’t get together into a boundle. Western blot for the expression of myosinVIIA showed that the mutations in MY07A led to the existence of a truncated protein. The Ik1 and ICa detected by electrophysiological were much higher in P-iPSC-derived hair cell-like cells. These results demonstrated that mutations in MY07A led to the disfunction of the hair cell through affecting the function of stereociliary bundles.Part three:To explore the possiblility of gene-corrected cell line in the use of clinic therapy, we corrected one of the compound heterozygous mutations in MYO7A in P-iPSCs through CRISPR-Cas9 mediated homologous recombination. Sequencing and restriction endonuclease detection indicated that 7.5±3.1% of the P-iPSCs were homozygously corrected. No off-target occurred and the corrected iPSCs (CP-iPSCs) still maintained the pluripotency. The CP-iPSCs could be induced to differentiate into hair cell-like cells expressing genes and proteins specific for hair cells and have the normal electrophysiological properties of hair cells. SEM detection showed the re-aggregation of stereociliary bundles and western blot exhibited the disappearance of the truncated protein. Moreover, the rate of cells labeled by FM1-43 reverted to the normal level, and the IK1, and Ica recorded in hair cell-like cells derived from CP-iPSCs reached the same level as that in hair cell-like cells derived from C- and CF-iPSCs. All these result demonstrated that one mutation of the heterozygous mutations in MYO7A was successfully corrected and thus the function of the hair cell like cells was restored.In summary, our study demonstrated that iPS cell could be generated from deafness patient and that the generated iPSCs could be induced to differentiate into inner ear hair cell-like cells. The gene correction of the deafness patient-derived iPSCs restored the function of the differentiated hair cell-like cells, which will be a new breakthrough for curing deafness.