Patent Document ID: 8778680
Application ID: 11966427
Patent Flag: 1

Claim One:
1. A method for the stable differentiation of Ntera2/D1 (NT2) cells into neuronal cells expressing Lmx1 b, Pitx3, tyrosine hydroxylase (TH) and Nurr1 in the absence of retinoic acid consisting essentially of the steps of: expanding NT2 cells in layer culture in the absence of retinoic acid; subculturing the expanded NT2 cells in the absence of retinoic acid; growing the subcultured NT2 cells in suspension culture in the absence of retinoic acid and exogenous growth factors for at least 11 days with the medium being changed daily; replating the cells on an adherent surface; growing the replated cells for at least 11 days; and assaying the replated cells to determine expression of mature neuronal markers Lmx1b, Pitx3, tyrosine hydroxylase, and Nurr1; wherein the replated cells become differentiated neuronal cells as determined by an increased expression of the mature neuronal markers; whereby the differentiated neuronal cells exhibit a stable neuronal phenotype for longer than 30 days after replating of cells.