Comparison of otic vesicle gene expression from E10.5 mouse and inner ear organoid cultures at 12 days-in-vitro with (BSFL) and without (BFL) TGFß inhibition

The early development of inner ear organoids—from germ layer to otocyst formation—is a highly controlled process with timed chemical cues that recapitulate major signals in vivo. We sought to elucidate additional morphogens that might guide this multi-stage process, improve efficiency, and influence cell fate decisions. Using a whole-transcriptomic approach with bulk RNASeq, we identified major differences in native otic vesicles (OVs) from E10.5 mice and stem cell-derived OVs from cultures at 12 days-in-vitro. Stem cell-derived samples included those treated with TGFß inhibitors at early stages of differentiation--to suppress mesendodermal fates--and those without TGFß inhibition, since other investigators identified this early inhibitory step as critical for organoid-genesis though dispensible for OV production. Differential gene expression between native and derived OVs identified factors related to anterior-posterior patterning as disregulated in the cell cultures, implicating aberrant retinoic acid (RA) signaling in the organoid culture paradigm. Overall design: RNASeq analysis of E10.5 mouse otic vesicles and otic vesicles isolated from inner ear organoid cell cultures treated with (BSFL samples) and without (BFL samples) TGFß inhibitors