Generation of Otic Lineages from Integration-Free Human-Induced Pluripotent Stem Cells Reprogrammed by mRNAs

Abstract

Damage to the sensory hair cells and the spiral ganglion neurons of the cochlea leads to deafness. Induced pluripotent stem cells (iPSCs) are a promising tool to regenerate the cells in the inner ear that have been affected by pathology or have been lost. To facilitate the clinical application of iPSCs, the reprogramming process should minimize the risk of introducing undesired genetic alterations while conferring the cells the capacity to differentiate into the desired cell type. Currently, reprogramming induced by synthetic mRNAs is considered to be one of the safest ways of inducing pluripotency, as the transgenes are transiently delivered into the cells without integrating into the genome. In this study, we explore the ability of integration-free human-induced pluripotent cell lines that were reprogrammed by mRNAs, to differentiate into otic progenitors and, subsequently, into hair cell and neuronal lineages. hiPSC lines were induced to differentiate by culturing them in the presence of fibroblast growth factors 3 and 10 (FGF3 and FGF10). Progenitors were identified by quantitative microscopy, based on the coexpression of otic markers PAX8, PAX2, FOXG1, and SOX2. Otic epithelial progenitors (OEPs) and otic neuroprogenitors (ONPs) were purified and allowed to differentiate further into hair cell-like cells and neurons. Lineages were characterised by immunocytochemistry and electrophysiology. Neuronal cells showed inward Na+ (INa) currents and outward (Ik) and inward K+ (IK1) currents while hair cell-like cells had inward IK1 and outward delayed rectifier K+ currents, characteristic of developing hair cells. We conclude that human-induced pluripotent cell lines that have been reprogrammed using nonintegrating mRNAs are capable to differentiate into otic cell types.

The Use Of Stem Cells In The Treatment Of Sensorineural Hearing Loss

“…Furthermore, these lines then successfully further differentiated into hair cell and neuronal lineages. The findings of this study indicate that mRNA-reprogrammed iPSCs are just as capable of producing otic lineages as lentivirus-induced ones while also being safer for clinical applications [27]. Overall, iPSCs in each study demonstrated that they are viable options for regenerating cells in the inner ear; however, more work still needs to be done to improve the survival and differentiation of iPSCs in the cochlea [24].…”

The Role of FoxG1 in the Inner Ear

“…Therefore, FoxG1 might affect the occurrence and development of age-related hearing loss by regulating multiple pathways. Foxg1 as one of the marker genes of inner ear progenitor cells and plays an important role in the process of inducing pluripotent stem cells to differentiate into inner ear cells ( Boddy et al, 2020 ). Therefore, the Foxg1 -related reprogramming technology has great application value to regenerate cells in the inner ear that are affected by pathology or damage.…”

A human induced pluripotent stem cell-based modular platform to challenge sensorineural hearing loss

“…Some of these protocols carried out completely in two‐dimensional (2D)‐cultures using treatments with a combination of small molecules and/or recombinant proteins to manipulate key signaling pathways of the inner ear development. The differentiation under 2D‐cultures can provide a relatively homogenous population of otic sensory progenitors and initial HC‐like cells in prolonged cell cultures 23‐26 . In contrast to 2D‐culture system, the induction of PSCs under three‐dimensional (3D)‐organoid strategy allows for the generation of multiple cell types and recapitulates, to some extent, in vivo composition of an inner ear sensory epithelium containing HC, supporting cell and sensory neuron‐like cells 27‐29 .…”