Methods for generating the inner ear and other cranial placode-derived tissues using pluripotent stem cells

A method for producing otic placode cells, the method comprising the steps of:

• (i) culturing pluripotent stem cells selected from the group consisting of human pluripotent stem cells and mouse pluripotent stem cells for between 1 to 2 days under conditions that result in formation of embryoid bodies from the cultured pluripotent stem cells;
• (ii) adding laminin and one or more extracellular matrix proteins selected from entactin, type IV collagen, fibronectin, vitronectin, and a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells to the embryoid bodies formed in step (i);
• (iii) culturing the embryoid bodies of step (ii) for about 2 days to about 3 days in the presence of BMP2, BMP4, or BMP7, and a TGFβ inhibitor to form non-neural ectoderm;
• (iv) culturing the non-neural ectoderm formed in (iii) for about 3 days to about 5 days in the absence of a BMP and the TGFβ inhibitor, and in the presence of an exogenous FGF and a BMP inhibitor, in floating culture, to generate a cell population comprising preplacodal ectoderm cells, wherein human preplacodal ectoderm cells express one or more of SIX1/4, EYA1/2, IRX1/2/3, AP2, DLX3/5/6, GATA2/3, FOXI1/3 and E-cadherin, or wherein mouse preplacodal ectoderm cells express at least one of Dlx3, Dlx5, Pax2, Pax8 and Eya1;
• and
• (v) culturing the preplacodal ectoderm cells formed in (iv), in floating culture, in the absence of the exogenous FGF and BMP inhibitor to obtain a cell population comprising otic placode cells, wherein human otic placode cells express one or more of Pax2, Pax8 and ECAD, or wherein mouse otic placode cells express one or more of Pax2, Pax8 and Sox2.

A method for producing inner ear sensory hair cells, the method comprising the steps of:

• (i) culturing pluripotent stem cells selected from the group consisting of human pluripotent stem cells and mouse pluripotent stem cells for between 1 to 2 days under conditions that result in formation of embryoid bodies from the cultured pluripotent stem cells;
• (ii) adding laminin and one or more extracellular matrix proteins selected from entactin, type IV collagen, fibronectin, vitronectin, and a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells to the embryoid bodies formed in step (i);
• (iii) culturing the embryoid bodies of step (ii) for about 2 days to about 3 days in the presence of BMP2, BMP4, or BMP7 and a TGFβ inhibitor to form non-neural ectoderm;
• (iv) culturing the non-neural ectoderm formed in (iii) for about 3 days to about 5 days in the absence of a BMP and the TGFβ inhibitor, and in the presence of an exogenous FGF and a BMP inhibitor, in floating culture, to generate preplacodal ectoderm, wherein human preplacodal ectoderm cells express one or more of SIX1/4, EYA1/2, IRX1/2/3, AP2, DLX3/5/6, GATA2/3, FOXI1/3 and E-cadherin, or wherein mouse preplacodal ectoderm cells express at least one of Dlx3, Dlx5, Pax2, Pax8 and Eya1;
• (v) culturing the preplacodal ectoderm cells formed in (iv), in floating culture, in the absence of the exogenous FGF and BMP inhibitor to obtain otic placode cells;
• and
• (vi) culturing the otic placode cells formed in (v) in floating culture in N2 medium comprising one or more extracellular matrix proteins for a time sufficient to form inner ear sensory hair cells, wherein human hair cells express MYO7A, BRN3C, and ATOH1, or wherein mouse hair cells express MYO7A.