Aisha Srinivasan
Research Mentor: Robert Duncan
Mentor Department: Kresge Hearing Research Institute, Medicine
Author(s): Aisha Srinivasan, Liqian Liu, R. Keith Duncan
Session: Session 6 (3:00 PM – 3:50 PM)
Presentation Type: Poster 42
Abstract
Inner ear organoids are three-dimensional, self-organizing tissue cultures derived from pluripotent stem cells that model the development of the normal mammalian inner ear. Organoid-genesis is a sensitive process that depends on precise timing of chemical cues to replicate major in vivo signals and developmental milestones. Translating well-established protocols from mouse to human stem cells has proven challenging. Human cultures are highly heterogeneous and require optimization for each cell line. One of the first milestones in this process, and thus one of the most sensitive steps for optimization, is the generation of otic (ear) placodal tissue from ectodermal germ layers. In vivo, the various lineages derived from the surface ectoderm are controlled in a dose-dependent manner by bone morphogenic protein (BMP). Increasing concentrations of BMP cause thinning of the ectodermal epithelium and systematically shift fate decisions from neuroectoderm to neural crest, placodes, and epidermis. Our research aims to identify the optimal concentration of BMP4 necessary to guide ectodermal tissue toward an otic fate, producing a higher yield of otic vesicle intermediates from which organoids will ultimately be derived. In this study, human induced pluripotent stem cell cultures were treated on culture day 0 with BMP4 at various doses (0.25 to 2.0 ng/ml). The thickness of the ectodermal epithelium was assessed on culture day 3. Published benchmarks suggest an optimal epithelial thickness of 25 to 50 µm for generation of otic placodes. As expected, we found a dose-dependent shift in thickness, with BMP4 concentrations of 1.5 to 2 ng/ml yielding average thickness of 23 to 29 µm. On culture day 25, when otic vesicles should be readily apparent, cryopreserved samples were stained with antibody markers to identify the presence of epithelial tissue (ECAD), placodal fates (SIX1 and SOX2), and otic specification (PAX2). We further characterized the morphology and efficiency of otic vesicle production for each BMP4 condition. Although quantification of these results continue, it appears that the efficiency of vesicle induction is maximal for BMP4 concentrations of 1.0 to 1.5 ng/ml. The majority of vesicles stained positively for ECAD, SIX1, and SOX2 but were PAX2 negative. Pax2 is a key marker in early development of otic vesicles, marking the transition from pre-placodal ectoderm to otic vesicles. The absence of this marker may indicate the vesicles are unspecified or represent other placodal lineages. This suggests that the current concentrations of BMP may be sufficient for placodal induction but insufficient for terminal otic specification. Establishing these optimal induction parameters is vital for the efficient generation of human ear organoids, which can facilitate downstream investigations into normal ear development, etiology of disease, therapeutic discovery, and regenerative medicine.
