Introduction Cholinergic system is one of the important systems of mammalian CNS. Cholinergic neurons distributed in brain and spinal cord and contributed to principal functions like: consciousness, learning and memory, and motor control. In this study we investigated the differentiation potentiality of mouse embryonic stem cells toward cholinergic neurons. The aim of this study was to evaluate the effect of sonic hedgehog (Shh), Retinoic Acid (RA), LIF, IL6 and NGF on differentiation of neural progenitor cells (NPCs), produced by lineage selection method, to cholinergic neurons. Material and methods Royan B1, mouse embryonic stem cells derived from C57BL/6 strain was used to produce aggregates. Aggregates were cultured in serum free medium to produce nestin positive or NPCs, then cell expansion was achieved by treatment with EGF and FGF2. Following withdrawal of EGF and FGF2, the cells were further cultured in presence or absence of differentiation factors in serum containing medium. Relative number of neurons and cholinergic neurons were assessed by immunohistochemical procedure using antibodies against MAP2, B-tubulin3, and ChAT. Results Data obtained show that around 70% of cells were B-tubulin3 positive. We found ChAT immunoreactivity in cultured cells in both treated and control groups. Conclusion This study shows that some of the neurons produced by lineage selection method are cholinergic neurons, and the percentage of cholinergic neurons increased after treatment by Shh, LIF and RA.

Keywords: Embryonic stem cells, Neuron, Cholinergic, Differentiation

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