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) TGFβ 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 TGFβ inhibitors at early stages of differentiation--to suppress mesendodermal fates--and those without TGFβ 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. 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) TGFβ inhibitors