Stable differentiation of NT2 cells

摘要

A method of differentiating adult stem cells, such as those derived from a teratocarcinoma cell line, the Ntera2/D1 clone (NT2). The developed cells exhibit a stable neurotransmitter phenotype without the required use of growth factors or retinoic acid in differentiation process, which may be difficult to completely remove during commercial production. An identification of specific neurotransmitters is possible in these differentiated NT2-derived neurons (NT2-N) after 30 days in culture or 30 days survival in vivo. The invention includes a method to stably differentiate neuronal stem/precursor cells to a neuronal phenotype for use in cell replacement therapy for neurodegenerative disease, stroke or spinal cord injury. At least four different types of neurons are produced from this method of differentiation: dopaminergic, cholinergic, GABAergic and glutaminergic. Additionally, since the cells are a cancer stem cell prior to differentiation, they may serve as a model system for developing anti-cancer therapies aimed at the cancer stem cell, rather than the more differentiated daughter cell.

主权项

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.