Single-Cell Fluorescence Analysis of Pseudotemporal Ordered Cells Provides Protein Expression Dynamics for Neuronal Differentiation

Abstract

Stem cell replacement therapy is a potential method for repopulating lost spiral ganglion neurons (SGNs) in the inner ear. Efficacy of cell replacement relies on proper differentiation. Defining the dynamic expression of different transcription factors essential for neuronal differentiation allows us to monitor the progress and determine when the protein functions in differentiating stem cell cultures. Using immortalized multipotent otic progenitors (iMOPs) as a cellular system for SGN differentiation, a method for determining dynamic protein expression from heterogeneous cultures was developed. iMOP-derived neurons were identified and ordered by increasing neurite lengths to create a pseudotime course that reflects the differentiation trajectory. The fluorescence intensities of transcription factors SOX2 and NEUROD1 from individual pseudotemporally ordered cells were measured. Individual cells were grouped by K-means clustering and the mean fluorescence intensity for each cluster determined. Curve fit of the mean fluorescence represented the protein expression dynamics in differentiating cells. The method provides information about protein expression dynamics in differentiating stem cell cultures.

Neurog1, Neurod1, and Atoh1 are essential for spiral ganglia, cochlear nuclei, and cochlear hair cell development

“…It promotes neuronal differentiation through the expression of Neurod1 68 . These findings suggest that Neurog1 can promote proliferation or neuronal differentiation and possibly impact hair cells without affecting cochlear nuclei 68 , 69 . It appears that a set of data support the transformation of astrocytes into neurons in Neurod1 70 and Neurog2 71 .…”

“…Quantification of the fluorescence intensity of nuclear proteins in immortalized multipotent otic progenitors showed expression dynamics of SOX2 and NEUROD1 from a progenitor into differentiated neurons. During neuronal differentiation, SOX2 levels decreased while NEUROD1 levels increased 69 . Evaluation of Neurog1 was excluded because of its dual roles in both proliferation and neuronal differentiation 68 .…”

“…The increase of Neurod1 expression is in line with what is known for Neurod1 in collaboration with Sox2 10 , 31 . Understanding the expression dynamics of crucial transcription factors helps design replacement strategies for lost sensory neurons 69 .…”

Age-Related Hearing Loss: Sensory and Neural Etiology and Their Interdependence

“…The development of SGNs is independent of both cochlear nuclei and cochlear hair cells (Rask-Andersen et al, 2005 ). Studying the growth and differentiation of SGNs could inform the steps needed to induce otic progenitors to replace lost neurons (Song et al, 2017 , 2019 ; Karlsson et al, 2019 ). SGN projections follow a simple tonotopic organization ( Figures 2G,H,H’ ): basal projection of the cochlea reaches the most dorsal part of the cochlear nuclei, whereas the apex reaches the ventral aspect of the cochlear nuclei (Muniak et al, 2016 ; Fritzsch et al, 2019 ).…”

Bioluminescence Imaging On-Chip Platforms for Non-Invasive High-Content Bioimaging

“…To date, fluorescence detection is the standard optical biosensing method used for cell analysis on-chip (Song et al, 2019;Vargas-Ordaz et al, 2021). Real-time fluorescence cell imaging and biomarker detection typically requires the need of end-point analysis, which is invasive and/or terminal, often involving complete sample sacrifice (Cullum et al, 2014;Wlodkowic et al, 2009).…”