Pluto Bioinformatics

GSE132635: Epigenetic and Transcriptional Regulation of Supporting Cell to Hair Cell Transdifferentiation

Bulk RNA sequencing

Purpose: We have recently extablished a new protocol for the generation of inner ear organoid or Lgr5-positive cochlear progenitors (LCPs). The purpose of this study is to analyze the expression pattern in these organoids as they differentiate and to compare them to native hair cells and supporting cells that were sorted from mouse inner ear at P2.; ; Methods: Lgr5-cochlear progenitors were harvested from newborn mice, dissociated and plated in Matrigel to generate organoids. The cells were expanded for 10 days and then differentiation for additional 10 days. Samples were harvested for RNA at D0 of differentiation, D2, D4 and D10 and RNA was extracted using RNeasy micro Kit (Qiagen). In parallel, whole cochleae were harvested from P2 litters of Atoh1-GFP, Lgr5-GFP and Sox2-GFP mice, sorted for GFP+ cells and RNA was extracted in the same manner. mRNA libraries were generated from using ThruPLEX DNA-seq kit (Rubicon Genomics) and then sequenced using Illumina NextSeq500 Single-End 75bp. Reads were aligned to mm10 and quality control was assessed using FastQC and Qualimap followed by FeatureCounts, Sailfish v0.10.1 and tximport. Differential expression was evaluated using DESeq2 and subsequent analysis was performed using Ingenuity Pathway Analysis Software (Qiagen) and standard R packages for RNA-Seq analysis. Samples were ran in duplicates and LCP samples were harvest from a single experiment for each of the duplicates.; ; Results: 40-50 million reads were detected per sample. PCA and Pearson correlation demonstrated a clear segragation of the samples to native cells Vs. cultured cells and along a gradient of differentiation. Multiple pairwise comparisons of differentially expressed genes were conducted to reveal several thousands of DE genes between the different LCP time points and between the native sorted cell types. Additional analysis of expression throughout differentiation segregated the genes into 8 clusters that represent proliferation, early differentiation and late differentiation gene groups in cochlear development. Clear downregulation of supporting cell and cell-cycle markers was detected as the cells differentiation alongside an upregulation of known hair cell markers. A similar distinction was detected between Atoh1-positive native hair cells and Lgr5-positive native supporting cells.; ; Conclusions: Our results validate the resemblance between our new organoid model system and native cochlear cells. It is evident that the cells at the end of the proliferation phase are similar to supporting cells (and not progenitor cells) and are therefore transdifferentiating into hair cells. The existence of several cochlear markers indicate a separation into outer hair cells and inner hair cells but does not exclude potential differentiation of a portion of the organoids towards a vestibule fate. SOURCE: Albert Edge (albert_edge@meei.harvard.edu) - Harvard Medical School