US 12,077,778 B2
Methods of generating human inner ear sensory epithelia and sensory neurons
Karl R. Koehler, Indianapolis, IN (US); and Eri Hashino, Indianapolis, IN (US)
Assigned to INDIANA UNIVERSITY RESEARCH AND TECHNOLOGY CORPORATION, Bloomington, IN (US)
Filed by INDIANA UNIVERSITY RESEARCH AND TECHNOLOGY CORPORATION, Indianapolis, IN (US)
Filed on Aug. 10, 2020, as Appl. No. 16/989,554.
Application 16/989,554 is a division of application No. 15/769,254, abandoned, previously published as PCT/US2016/058121, filed on Oct. 21, 2016.
Claims priority of provisional application 62/244,568, filed on Oct. 21, 2015.
Prior Publication US 2020/0370007 A1, Nov. 26, 2020
Int. Cl. C12N 5/0793 (2010.01); C12N 5/0735 (2010.01); C12N 5/074 (2010.01)
CPC C12N 5/062 (2013.01) [C12N 5/0606 (2013.01); C12N 5/0607 (2013.01); C12N 2501/115 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/415 (2013.01); C12N 2506/02 (2013.01); C12N 2533/52 (2013.01); C12N 2533/90 (2013.01)] 20 Claims
OG exemplary drawing
 
1. A method of obtaining a three-dimensional composition comprising human inner ear sensory tissue, the method comprising the steps of:
(a) culturing human pluripotent stem cell aggregates in a culture medium comprising Fibroblast Growth Factor-2 (FGF-2), a Bone Morphogenetic Protein (BMP), and a small molecule inhibitor of Transforming Growth Factor Beta (TGFB)-mediated signaling for four days;
(b) further culturing the cultured aggregates of (a) in the presence of a Fibroblast Growth Factor (FGF) and an inhibitor of BMP signaling for four days;
(c) contacting the further cultured aggregates of (b) to a Wnt agonist for four days, whereby cells within the contacted aggregates differentiate into pre-otic epithelial cells;
(d) embedding the pre-otic epithelial cells obtained in step (c) in droplets of a semi-solid medium comprising extracellular matrix;
(e) culturing the droplets of embedded pre-otic epithelial cells in a static culture in the presence of a Wnt agonist for 6 days under conditions that promote self-assembly of embedded pre-otic epithelial cells into otic vesicles; and
(f) culturing the droplets of embedded pre-otic epithelial cells of (e) further in a stirring culture for at least 22-42 days, whereby a three-dimensional composition comprising human inner ear sensory tissue is obtained.