Effect Of Genetic Correction Of Deafness- Causing MYO15A Mutation In Human IPSCs On Morphology And Function Of The Derived Hair Cell-like Cells

Deafness and hearing loss is one of the major health problems worldwide. Human ear is responsible for hearing and balance, and the inner ear hair cells in ear are key cells for auditory sensory. When sound waves pass, stereocilia on the surface of hair cells will swing, which switch the acoustic signals into electrical signals and result in hearing. Hair cell damage or missing is the main cause evocating deafness and hearing loss. Noise, aging, genetic mutations or drug abuse (especially aminoglycoside antibiotics) can lead to hair cell damage or missing. In mammals including human beings, inner ear hair cells cannot be regenerated any more once they are damaged, which result in permanent deafness. Exploring how to regenerate hair cells in human beings will be helpful for curing deafness or hearing loss.Induced pluripotent stem cells (iPSCs) are similar to embryonic stem cells (ESCs). The advantages of no ethical problems, pluripotency, infinite amplification, patient-specificity, and no immune rejection effect make iPSCs exhibit broad application prospect in regenerative medicine, disease modeling and drug screening fields. The appearing of gene editing technology, especially the rapid development of the CRJSPR/Cas9 technology makes human beings can easily manipulate or correct gene. In recent years, breakthrough in the study of inner ear hair cell regeneration makes hair cell regeneration much easier. In this study, we established an iPSC line with a deafness-causing MYO15A mutation; by CRISPR/Cas9 technology, we genetically corrected the MYO15A mutation in M-/-iPSCs and induced iPSCs differentiation towards hair cells. We revealed that the genetic correction of MYO15A reversed the morphological and functional defects in hair-like cells causing by MYO15A mutation, and established foundation for the effective treatment of deafness.Part I:By retrovirus, we generated three iPSC lines (M-/-iPSC, M+/iPSC and M+/+iPSC) from a deafness patient with MY015A mutation (MY015A c.4642G>A, c.8374G>A), his father (MYO15A c.8374G>A) and a female girl with normal hearing function. Three iPSC lines exhibited characteristics similar to ESC, such as the AP-positivity, the positive expression of pluripotent stem cell-specific marker proteins OCT4, SOX2, NANOG, SSEA4, TRA-1-60 and TRA-1-81, and the potentials of tridermogenesis in vitro and vivo. By karyotype analysis, we found that the iPSC lines maintained a normal karyotype.Part II:Through monolayer layer culture method, we induced the three iPSC lines into otic progenitors. The otic progenitors expressed otic-ralated marker proteins and genes. By co-culture of otic progenitors with chicken utricle stromal cells, we successfully generated hair cell-like cells from the three iPSC lines. These hair-like cells expressed hair cell specific marker genes and proteins, uptook specifically FM1-43 dye, and displayed inner ear hair cell-like potassium current and calcium current. In addition, the hair cell-like cells developed microvilli-based stereocilia-like structures revealed by scanning electronic microscope. However, we observed the effect of MYO15A mutation on morphology and function of the derived hair cell-like cells. Compared with M+/+iPSCs and M+/iPSCs, the hair cell-like cell generated from M-/’iPSCs exhibited morphology and function defects, like actin disorder and significantly shorter stereocilia.Part III:By CRISPR-Cas9 mediated homologous recombination technology, we genetically corrected the MYO15A c.4642G>A mutation in M-/-iPSCs. We designed and constructed the targeting vector pX330-maxGFP-sgRNA4, homologous recombination template ssODN and pUC19-MYO15Asynonymous. By flow cell sorting and Sanger sequencing, we screened the genetically corrected MC/-iPSC cell line. MC/- iPSCs maintained the normal karyotype, expressed pluripotent stem cell marker genes and proteins, and possessed the potentials of tridermogenesis in vitro and vivo.Part IV:To understand the effect of genetic correction of MYO15A mutation in human M-/-iPSCs on morphology and function of the derived hair cell-like cells, we induced the M/-iPSCs into inner ear hair cell-like cells. Similar to M+/+iPSCs and M+/-iPSCs, the otic progenitors from MC/-iPSCs expressed otic-related genes and proteins, hair cell-like cells expressed hair cell specific marker genes and proteins. Hair cell-like cells from MC/-iPSCs also displayed similar electrophysiological current density with hair cell-like cells from M+/+iPSCs and M+/-iPSCs. In addition, these cells displayed the normal actin filament organization, the normal stereocilia length, which was significantly different from hair cell-like cells fromM-/-’iPSCs (actin disorder and significantly shorter stereocilia). Our research showed that the genetic correction of MYO15A reversed the morphological and functional defects in hair-like cells causing by MY015A mutation,.In conclusion, through genetic correction of MYO15A in M-/-iPSCs and generation of hair cell-like cells, we demonstrated the feasibility of generating inner ear hair cells from human iPSCs and the functional rescue of deafness-causing gene mutations using genetic correction.