Retinoic acid induces cholinergic differentiation of NTera 2 human embryonal carcinoma cells

Retinoic acid (RA), a natural metabolite of vitamin A, influences the survival and neurotransmitter phenotype of several classes of vertebrate neurons during development. We now report that RA induces a subpopulation of NTera 2/clone D1 (NT2) human embryonal carcinoma cells to differentiate into postmitotic cells with cholinergic properties (NT2-N cells). After growth for 6 days in the presence of RA (10 microM) low levels of the acetylcholine-synthesizing enzyme choline acetyltransferase (ChAT) were detected in NT2 cell cultures. ChAT activity in the NT2 cell cultures continued to increase for at least an additional 22 days to a final activity of 50 pmol ACh synthesized/min/mg protein. Immunohistochemical staining of RA-treated cultures demonstrated that only those cells with a neuronal morphology (NT2-N cells) expressed the human ChAT protein. Since such cells comprised a small proportion (approximately 20%) of the population, the ChAT activity per neuronal cell was estimated to approach 250-300 pmol ACh/min/mg protein. Cultures composed of > 95% NT2-N cells had significantly lower ChAT specific activities and this could be increased by either ciliary neurotrophic factor or leukemia inhibitory factor, but not by nerve growth factor. We conclude that NT2 cells provide a system in which to study the molecular events that underlie neurotransmitter choice during the differentiation of human cholinergic neurons.