Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells.

Authors:
Koehler KR, Nie J1, Longworth-Mills E, Liu XP, Lee J, Holt JR, Hashino E.
In:
Source: Nat Biotechnol
Publication Date: (2017)
Issue: 35(6): 583-589
Research Area:
Dermatology/Tissue Engineering
Gene Expression
Cells used in publication:
Embryonic Stem Cell (ES), human
Species: human
Tissue Origin: embryo
Platform:
4D-Nucleofector™ X-Unit
Experiment
To construct the donor vector, a 2A-eGFP-PGK-Puro cassette (Addgene #31938)39 flanked by two 1kb homology arms PCR amplified from extracted WA25 hESC genomic DNA were cloned into a pUC19 backbone. The two gRNA vectors and the donor vector, as well as a vector expressing Cas9n under the control of CMV promoter (Addgene #41816)40 were transfected into WA25 hESCs with 4D Nucleofector (Lonza) using the P3 Primary Cell 4D-Nucleofector X kit and Program CB-150. After nucleofection, cells were plated in growth medium containing 1× RevitaCell (Thermo Fisher) for improved cell survival rate, and 1µM of Scr7 (Xcessbio) for higher HDR efficiency41. 0.5 µg ml-1 puromycin selection was performed for 10 days starting from 48 hours post-nucleofection. The PGK-Puro sub-cassette flanked by two LoxP sites was removed from the genome after puromycin selection by nucleofection of a Cre recombinase expressing vector (Addgene #13775). Clonal cell lines were established by low-density seeding (1 – 3 cells cm-2) of dissociated single hESCs followed by isolation of hESC colonies after 5-7 days of expansion.
Abstract
The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate. Over 2 months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR-Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system should facilitate the study of human inner ear development and research on therapies for diseases of the inner ear.