Transcription factor induced conversion of human fibroblasts towards the hair cell lineage

Abstract

Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain. Although some spontaneous HC regeneration has been observed in neonatal mammals, the very small pool of putative progenitor cells that have been identified in the adult mammalian cochlea is not able to replace the damaged HCs, making any hearing impairment permanent. To date, guided differentiation of human cells to HC-like cells has only been achieved using either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, use of such cell types suffers from a number of important disadvantages, such as the risk of tumourigenicity if transplanted into the host´s tissue. We have obtained cells expressing hair cell markers from cultures of human fibroblasts by overexpression of GFI1, Pou4f3 and ATOH1 (GPA), three genes that are known to play a critical role in the development of HCs. Immunocytochemical, qPCR and RNAseq analyses demonstrate the expression of genes typically expressed by HCs in the transdifferentiated cells. Our protocol represents a much faster approach than the methods applied to ESCs and iPSCs and validates the combination of GPA as a set of genes whose activation leads to the direct conversion of human somatic cells towards the hair cell lineage. Our observations are expected to contribute to the development of future therapies aimed at the regeneration of the auditory organ and the restoration of hearing.

Atoh1 is repurposed from neuronal to hair cell determinant by Gfi1 acting as a coactivator without redistribution of its genomic binding sites

“…Together, these observations suggest that Pou4f3 establishes lineage-specific competence for Atoh1-driven sensory cell differentiation, perhaps as a pioneer factor that allows Atoh1 access to its sensory targets. Consistent with this are reports that Pou domain TFs can reprogram mouse and human fibroblasts into iHC (Duran Alonso et al 2018). Interestingly, this implies that Atoh1 is not a pioneer factor in HC differentiation in vitro (and perhaps in vivo).…”

Stepwise Induction of Inner Ear Hair Cells From Mouse Embryonic Fibroblasts via Mesenchymal- to-Epithelial Transition and Formation of Otic Epithelial Cells

“…In this way, we were able to achieve induction of inner ear HCs that express multiple HC markers and acquire cilia and functions. Although overexpression of GFI1, Pou4f3, and ATOH1 together has been previously shown to induce conversion of human fibroblasts toward the HC lineage, that approach only allowed generation of cells expressing HC markers but failed to show their functions (Duran Alonso et al, 2018). In our experiments, following the reprogramming of MEFs to epithelial-like cells, transient overexpression of these three TFs in MEF-derived epithelial-like cells with Dox treatment at days 7-14 induced a lineage conversion of epithelial-like cells to OECs (Figure 3).…”

Multifaceted Actions of GFI1 and GFI1B in Hematopoietic Stem Cell Self-Renewal and Lineage Commitment

“…and has been shown to physically interact with the former three to target many hair cell regulators (Li et al, 2020). Either with or without SIX1, primary mouse embryonic and even adult human fibroblasts could be successfully converted into cells that exhibited most of the physiological traits of inner ear hair cells opening new avenues in potential treatment of deafness caused by the loss of these sensory cells (Figure 4; Duran Alonso et al, 2018;Menendez et al, 2020).…”

“…Strategies to generate induced hair cell-like cells through trans-differentiation of non-hair cells in vitro by forced expression of GFI1 in combination with other factors. The efficiency to produce cells that harbor hair cell characteristics in vitro is indicated along the starting cell type used to achieve trans-differentiation Duran Alonso et al, 2018;Menendez et al, 2020).…”