The method provides information about protein expression dynamics in differentiating stem cell cultures. Curve fit of the mean fluorescence represented the protein expression dynamics in differentiating cells. Individual cells were grouped by K-means clustering and the mean fluorescence intensity for each cluster determined. The fluorescence intensities of transcription factors SOX2 and NEUROD1 from individual pseudotemporally ordered cells were measured. iMOP-derived neurons were identified and ordered by increasing neurite lengths to create a pseudotime course that reflects the differentiation trajectory. 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. 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. Efficacy of cell replacement relies on proper differentiation. Stem cell replacement therapy is a potential method for repopulating lost spiral ganglion neurons (SGNs) in the inner ear. 2Stem Cell Research Center and Keck Center for Collaborative Neuroscience, Rutgers University, Piscataway, NJ, United States.1Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ, United States.Laureano 1,2†, Kishan Patel 1, Sylvia Yip 1, Azadeh Jadali 1,2 and Kelvin Y.
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