Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs results in severe hearing impairment. Damaged HCs can be regenerated by neighboring supporting cells (SCs), thus the functional regeneration of HCs is the main goal for the restoration of auditory function in vivo. Here, cochlear SC trans-differentiation into outer and inner HC by the induced expression of the key transcription factors Atoh1 and its co-regulators Gfi1, Pou4f3, and Six1 (GPAS), which are necessary for SCs that are destined for HC development and maturation via the AAV-ie targeting the inner ear stem cells are successfully achieved. Single-cell nuclear sequencing and lineaging tracing results showed that the majority of new Atoh1-derived HCs are in a state of initiating differentiation, while GP (Gfi1, Pou4f3) and GPS (Gfi1, Pou4f3, and Six1) enhanced the Atoh1-induced new HCs into inner and outer HCs. Moreover, the patch-clamp analysis indicated that newborn inner HCs induced by GPAS forced expression have similar electrophysiological characteristics to those of native inner HCs. Also, GPAS can induce HC regeneration in the HC-damaged mice model. In summary, the study demonstrates that AAV-mediated co-regulation of multiple genes, such as GPAS, is an effective means to achieve functional HC regeneration in the mouse cochlea. 

AAV‐mediated Gene Cocktails Enhance Supporting Cell Reprogramming and Hair Cell Regeneration

Mechanosensory hair cells of the mature mammalian organ of Corti do not regenerate; consequently, loss of hair cells leads to permanent hearing loss. Although nonmammalian vertebrates can regenerate hair cells from neighboring supporting cells, many humans with severe hearing loss lack both hair cells and supporting cells, with the organ of Corti being replaced by a flat epithelium of nonsensory cells. To determine whether the mature cochlea can produce hair cells in vivo, we reprogrammed nonsensory cells adjacent to the organ of Corti with three hair cell transcription factors: Gfi1, Atoh1, and Pou4f3. We generated numerous hair cell-like cells in nonsensory regions of the cochlea and new hair cells continued to be added over a period of 9 wk. Significantly, cells adjacent to reprogrammed hair cells expressed markers of supporting cells, suggesting that transcription factor reprogramming of nonsensory cochlear cells in adult animals can generate mosaics of sensory cells like those seen in the organ of Corti. Generating such sensory mosaics by reprogramming may represent a potential strategy for hearing restoration in humans.

Expression of Atoh1, Gfi1, and Pou4f3 in the mature cochlea reprograms nonsensory cells into hair cells.

Abstract Controlling stem cell differentiation is a longstanding goal in biomedical research. Here we explore how cell fate is influenced by RNA condensates, specifically P-bodies, which modulate gene expression posttranscriptionally. We profiled the transcriptomes of biomolecular condensates in diverse developmental contexts spanning multiple vertebrate species. Our analyses revealed conserved, cell type-specific sequestration of untranslated RNAs encoding cell fate regulators. P-body RNA contents do not reflect active gene expression in each cell type but are enriched for translationally repressed transcripts characteristic of the preceding developmental stage. Mechanistically, P-body contents are controlled by microRNAs and can be profoundly reshaped by perturbing AGO2 or polyadenylation site usage. Applying these insights to stem cell differentiation, we show that manipulating P-body assembly or microRNA activity can direct naive mouse and human pluripotent stem cells toward totipotency or primed human embryonic cells toward the germ cell lineage. Our findings link cell fate decisions to RNA condensates across vertebrates and provide a means of controlling cell identity. 

Selective RNA sequestration in biomolecular condensates directs cell fate transitions

Sensory hair cell loss in the vestibular organs of the inner ear causes balance disorders which are essentially irreversible due to the lack of significant hair cell regeneration. Here, we administered a γ-secretase inhibitor to an adult mouse model of vestibular hair cell loss. The treatment resulted in complete regeneration of type II and partial restoration of type I hair cells after one month and restored vestibuloocular reflexes across all frequencies of rotational and linear acceleration. Further recovery was apparent at 8 months. Genetic deletion of Notch1 in supporting cells identified Notch1 as the target of the drug. The results demonstrate that a single injection of a γ-secretase inhibitor is a viable therapy for functional restoration of the vestibular system in patients with balance disorders. 

Regeneration of Sensory Hair Cells and Restoration of Vestibular Function by Notch Inhibition

Abstract Cochlear hair cells can be killed by loud noises, ototoxic drugs, and natural aging. Once lost, mammalian hair cells do not naturally regenerate, leading to permanent hearing loss. Since the mammalian cochlea lacks any intrinsic ability to regenerate, genetic reprogramming of cochlear supporting cells that lie adjacent to hair cells is a potential option for hearing restoration therapies. We targeted cochlear supporting cells with three hair cell transcription factors: Atoh1, or Atoh1 + Gfi1, or Atoh1 + Gfi1 + Pou4f3 and found that 1- and 2-factor reprogramming is not sufficient to reprogram adult supporting cells into hair cells. However, activation of all three hair cell transcription factors reprogrammed some adult supporting cells into hair cell-like cells. We found that killing endogenous hair cells significantly improved the ability of supporting cells to be reprogrammed and regenerated numerous hair cell-like cells throughout the length of the cochlea. These regenerated hair cell-like cells expressed myosin VIIa and parvalbumin, as well as the mature outer hair cell protein prestin, were innervated, expressed proteins associated with ribbon synapses, and formed rudimentary stereociliary bundles. Finally, we demonstrate that supporting cells remained responsive to transcription factor reprogramming for at least 6 weeks after hair cell damage, suggesting that hair cell reprogramming may be effective in the chronically deafened cochlea. 

Reprogramming with Atoh1, Gfi1, and Pou4f3 promotes hair cell regeneration in the adult organ of Corti

Recent development of RNA velocity uses master equations to establish the kinetics of the life cycle of RNAs from nascent RNA to mature RNA to degradation. To feed this kinetic analysis, simultaneous measurement of nascent RNA and mature RNA in single cells is greatly desired. However, the majority of single-cell RNA-seq chemistry only captures mature RNA species to measure gene expressions. Here, we develop a one-step total-RNA chemistry-based scRNA-seq method: snapTotal-seq. We benchmarked this method with multiple single-cell RNA-seq assays in their performance in kinetic analysis of cell cycle by RNA velocity. Next, with LASSO regression between transcription factors, we identified the critical regulatory hubs mediating the cell cycle dynamics. We also applied snapTotal-seq to profile the oncogene-induced senescence and identified the key regulatory hubs governing the entry of senescence. Furthermore, from the comparative analysis of nascent RNA and mature RNA, we identified a significant portion of genes whose expression changes occurred in mature RNA but not to the same degree in nascent RNA, indicating these gene expression changes are mainly controlled by post-transcriptional regulation. Overall, we demonstrate that snapTotal-seq can provide enriched information about gene regulation, especially during the transition between cell states.

Single-cell Total-RNA Profiling Unveils Regulatory Hubs of Transcription Factors

Spontaneous DNA damage frequently occurs on the human genome, and it could alter gene expression by inducing mutagenesis or epigenetic changes. Therefore, it is highly desired to profile DNA damage distribution on the human genome and identify the genes that are prone to DNA damage. Here, we present a novel single-cell whole-genome amplification method which employs linear-copying followed by a split-amplification scheme, to efficiently remove amplification errors and achieve accurate detection of DNA damage in individual cells. In comparison to previous methods that measure DNA damage, our method uses a next-generation sequencing platform to detect misincorporated bases derived from spontaneous DNA damage with single-cell resolution.

Single-cell Damagenome Profiling by Linear Copying and Splitting based Whole Genome Amplification (LCS-WGA).

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Expression of Atoh1, Gfi1, and Pou4f3 in the mature cochlea reprograms nonsensory cells into hair cells.

Single-cell Total-RNA Profiling Unveils Regulatory Hubs of Transcription Factors

Single-cell Damagenome Profiling by Linear Copying and Splitting based Whole Genome Amplification (LCS-WGA).