Research Assistant, Graduate Student Boston Children's Hospital, Harvard Medical School Boston, Massachusetts, United States
Abstract: Human inner ear organoids (IEOs) derived from human embryonic or induced pluripotent stem cells provide a powerful model for studying inner ear development. These in vitro models closely mimic in vivo otic vesicle formation and supporting tissue development. However, the generation of "on-target" otic sensory epithelium remains quantitatively limited and deeply embedded within 3D aggregates, reducing their utility for disease modeling and therapeutic applications.
Here, we systematically optimized human IEO differentiation following key developmental principles. First, we fine-tuned BMP4 concentrations at Day 0 to bias 3D stem cell aggregates toward a preplacodal ectoderm (PPE) fate, a prerequisite for otic lineage commitment. Recent studies have highlighted the critical role of exogenous BMP4 and WNT signaling in otic specification, with early WNT activation shown to improve otic induction efficiency in mouse IEOs.
Building on this, we hypothesized that earlier WNT activation in BMP4-optimized human IEOs would enhance otic progenitor formation. To test this, we analyzed morphological changes (Days 0-30) using a newly generated dual-reporter hiPSC line to enable real-time tracking of otic (OC90-mRuby3) and sensory (SOX2-GFP) differentiation. While early WNT signaling (Day 4) significantly promoted otic vesicle formation, it significantly reduced SOX2+ signals in the otic epithelium, suggesting over-dorsalization and disruption of sensory lineage specification.
Given that SOX2+ sensory epithelium arises ventrally in the otic vesicle in vivo, requiring both WNT and Shh signaling for proper maturation, we hypothesized that Shh activation following otic vesicle formation could restore sensory lineage development. Using live-cell imaging, we found the treatment with the Shh agonist Purmorphamine successfully restored SOX2+ sensory epithelium.
Our findings demonstrate that precise modulation of WNT and Shh signaling can significantly influence the sensory epithelium development in vitro. These optimized human IEOs not only improve differentiation efficiency but also facilitate the isolation and enrichment of sensory otic tissues, paving the way for future applications on inner ear disease modeling, drug screening, and regenerative medicine.
