Abstract
Damage to the mechanosensory hair cells of the inner ear causes permanent hearing loss in millions of people each year. Although some vertebrate species can regenerate hair cells, mammals lack any regenerative capacity. A core goal of the hearing research field is to efficiently stimulate hair cell regeneration in mammals to prevent hearing loss. In recent years, many crucial factors that play a role in hair cell development have been discovered. The transcription factor Atoh1 is known to be necessary and sufficient for hair cell development in embryonic mouse inner ear and thus represents the standard in hair cell regeneration strategies. However, evidence suggests that overexpression of Atoh1 alone is unable to drive hair cell-like cell transformation in vitro, suggesting that additional factors may be required. Multiple cell type-specific factors, including microRNAs (miRNAs) and transcription factors (TFs) that regulate gene expression, have been used in prior studies to drive cell transformation in iPSC and neuronal cell generation. Specific miRNAs have been shown to be required for hair cell development including, miRNA-183 family members (miR-183, miR-96, and miR-182). Additionally, other transcription factors including Pou4f3 and Gfi1 are necessary for hair cell maintenance and survival. Our goal is to test whether a combination of hair cell TFs and miRNAs can generate hair cell-like cells more efficiently than Atoh1 alone. To ultimately examine this question, our immediate objective was to construct and validate an array of expression vectors that deliver various combinations of Atoh1, Pou4f3, Gfi1, and the miRNA-183 family. A novel expression vector was previously developed that would deliver both hair cell miRNAs and TFs from a single transcript. The transcript produces miRNA-183 family members from an intron and individual transcription factors (Atoh1, Pou4f3, and Gfi1) and red fluorescent protein from a single open reading frame by ribosome-mediated cleavage of intervening viral 2A peptide elements. From this parent vector we derived a series of eight plasmid vectors that deliver different combinations of the hair cell factors to ultimately assess which factors enhance the ability of Atoh1 to promote hair cell-like cell differentiation. Plasmid vectors were transfected into mouse otic precursor cells and general protein expression was confirmed by visualization of RFP fluorescence, which demonstrated decreased levels of plasmid expression in transfections of larger constructs. Transfected cell total RNA was analyzed via qRT-PCR methodologies and validated increased levels of miRNA-183 family detection. Six plasmid vectors were converted to Adenovirus vectors to develop a more efficient delivery method. Adenoviral expression efficiency was validated by both visualization of RFP fluorescence and subsequent flow cytometric analysis of infected HEK293 cells, which confirmed earlier observations of decreased expression efficiency of larger constructs. qRT-PCR analysis of infected cell total RNA further verified miRNA-183 family member processing. Detection of TFs via western blot analysis indicated protein processing was occurring, although cleavage at one 2A peptide appeared to be relatively inefficient. Phalloidin staining of transfected mouse otic precursor cells did not reveal noticeable morphological changes in actin structure after 48 hours. However, the construction and validation of these vectors will offer valuable tools for studying the combined effects of hair cell factors in various cell culture models. A successful combination of such factors that improves hair cell differentiation will facilitate in vitro study of hair cell development and is likely to enhance regenerative strategies for hair cell replacement and hearing restoration.