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.
Highlights
Hearing loss is the most prevalent sensorineural deficit in humans, most frequently caused by damage of hair cells (HCs)
Overexpression of the three genes was confirmed by Quantitative reverse transcription polymerase chain reaction (qPCR) and their coexpression in the transduced cells was analyzed by immunocytochemistry
To assess conversion towards the HC lineage we examined the expression of the HC markers MYOSIN VIIA and ESPIN, typically used for this purpose, together with endogenous expression of POU4F3 via qPCR
Summary
Hearing loss is the most prevalent sensorineural deficit in humans, most frequently caused by damage of hair cells (HCs). A promising alternative to the ESCs or iPSCs may be the use of patient-derived somatic cells that, fully differentiated, can be changed into the desired cell fate while bypassing the full reprogramming process typically undergone by iPSCs [15,16,17,18,19] This strategy has already resulted in the successful transdifferentiation of fibroblasts, hepatocytes, astrocytes and various differentiated blood cell types into other cell lineages (e.g. cardiomyocytes, neurons, macrophages) [20,21,22,23,24,25,26]. Epigenetic alterations have been shown to occur following exposure of the cells to chromatin modifiers such as demethylating agents and histone deacetylase inhibitors, or overexpression of transcription factors such as Sox-2 [31] and Oct-4 [24,32] This results in the transient expression of sets of genes that are associated with a variety of cell lineages. This is the first time that conversion of human somatic cells towards the hair cell lineage is reported
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