Inner ear organoids derived from human pluripotent stem cells: comparisons to human foetal inner ear

Mattei C1,2, Lim R3, Drury HR3, Tadros MA3, Nasr B1,4, Chatterton R1,2, Kulkarni T1,2, Jamshidi P1,2, D'Abaco G2, Nayagam BA5 and Dottori M1,2,6

  1. Centre for Neural Engineering, Melbourne School of Engineering, University of Melbourne, Victoria, Australia.
  2. Department of Biomedical Engineering, Melbourne School of Engineering, University of Melbourne, Victoria, Australia.
  3. School of Biomedical Sciences and Pharmacy, Faculty of Health and Medicine, University of Newcastle, NSW, Australia.
  4. Department of Electrical and Electronic Engineering, Melbourne School of Engineering, University of Melbourne, Victoria, Australia.
  5. Departments of Audiology and Speech, Pathology & Ophthalmology, University of Melbourne, Victoria, Australia.
  6. Illawarra Health and Medical Research Institute, University of Wollongong, NSW, Australia.

Introduction: In mammals, sensory hair cells do not regenerate. Consequently, the derivation of inner ear tissue from human pluripotent stem cells (hPSC) offers an opportunity to study human inner ear development and provides a platform for drug screening and disease modelling. Methods: A dynamic three dimensional rotary cell culture system was used to derive inner ear organoids from human PSCs for 16 weeks in-vitro. Differentiation and mechanosensitiviy of hPSCs-derived organoids were examined using a combination of qPCR, immunofluorescent labelling, and AM142 staining. Helium microscopy and electrophysiology compared the anatomical and physiological characteristics of inner ear organoids to foetal human inner ear. Results: Inner ear organoids show temporal expression of key developmental hair cell markers including Pax2, Atoh1, MyosinVIIa, and CtBP2 by immunofluorescence and qPCR. AM143 fluorescence in organoid cells is indicative of mechanosensitivity. Cells have outward currents (350 pA to 5 nA) consistent with developing human type II vestibular hair cells (12-16 weeks gestation). A subset of oganoid cells also have sodium currents. Striking morphological similarities were detected between inner ear organoids and developing inner ear using helium microscopy. Conclusion: We describe a novel three dimensional system for modelling human inner ear development using rotary cell culture. Preliminary data suggests this system is capable of generating a population of inner ear hair cells which resemble an early vestibular phenotype.