A simple method for differentiation of H9 cells into neuroectoderm.

Human embryonic stem cells (ESCs) can form neuroectoderm (NE), providing a platform for in vitro dissection of NE formation. However, human ESCs can differentiate into all three germ layers. It thus is crucial to develop efficient methods for differentiation of human ESCs into NE cells. Both plating cell density and localized cell density (LCD) affect NE differentiation. Here, we developed a cell cluster-based NE differentiation method, in which both plating cell density and LCD are under control. Using our new method, high plating cell densities promote expression of PAX6, a NE marker protein. Two SMAD signaling blockers, SB431542 and NOGGIN, downregulate OCT4 and upregulate PAX6, while does not affect mRNA expression of GATA2 after 5 d of differentiation. Moreover, IB analysis showed a time-dependent upregulation of PAX6 and beta-III-tubulin together with a downregulation of OCT4 during the neural differentiation. Coexpression of both TH and beta-III-tubulin in the H9-derived cells was also detected, proving the NE cells have an ability to differentiate into one of the specific neurons. Together, we established a simple method for generating NE cells from H9 cells, which might contribute to develop high efficient method for neural differentiation.