Methods for generating the inner ear and other cranial placode-derived tissues using pluripotent stem cells
Disclosed herein are methods and compositions for generating cultures and isolated cell populations containing preplacodal ectoderms cells, otic placode cells, and inner ear sensory hair cells derived from pluripotent cells by modulating TGFβ, BMP, and FGF signaling pathways under defined culture conditions. Also described are methods for obtaining non-otic placodal tissues from pluripotent stem cells. Methods for identifying agents that induce or enhance differentiation and generation of hair cells are also disclosed. Methods for identifying cytoprotective agents for hair cells are also described.
1. A method for producing preplacodal ectoderm 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 one of (1) a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells or (2) an effective amount of laminin and entactin, wherein the effective amount facilitates the formation of a basement membrane, to the cultured embryoid bodies formed in step (i); and (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 TGF-β inhibitor to form non-neural ectoderm cells; and (iv) culturing the non-neural ectoderm cells formed in (iii) for about 3 days to about 5 days in the absence of the BMP2, BMP4, or BMP7 and the TGF-β inhibitor, and in the presence of an exogenous FGF and a BMP inhibitor, in floating culture for about 4 days to about 6 days after initiation of step (i), to generate a cell population comprising human or mouse preplacodal ectoderm cells, wherein said 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 said mouse preplacodal ectoderm cells express at least one of Dlx3, Dlx5, Pax2, Pax8 and Eya1.
2. The method of claim 1 , wherein step (iii) comprises culturing the embryoid bodies in the presence of BMP4.
3. The method of claim 1 , wherein the pluripotent stem cells are human pluripotent stem cells.
4. The method of claim 3 , wherein the BMP2, BMP4, or BMP7, and the TGFβ inhibitor are added to the embryoid bodies formed in step (i) on day 5, 6, or 7.
5. The method of claim 1 , wherein the gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells or an effective amount of laminin and entactin is added to the cultured embryoid bodies on day 1.