Abstract
ABSTRACT Aim: Human embryonic stem cell (hESC)-derived motor neurons (MNs) can model neurodegenerative diseases, in other words, for the development of cell-based drug screening assays. However, efficient MN generation is challenging and high level of contamination of undifferentiated cells remains a significant problem when culturing hESC-derived MNs. We describe a protocol for the removal of undifferentiated cells using the DNA synthesis inhibitor arabinofuranosyl cytidine (Ara-C). Materials & methods: hESC-MNs were treated with Ara-C after the last step of differentiation. FACS analysis and fluorescence microscopy were used to identify and quantify MNs. Results: HB9/ChAT-positive live mature MNs were enriched 3.9-times in Ara-C-treated cultures when compared with untreated cells. The Ara-C-treated MNs are electrophysiologically functional and discharge action potentials. Conclusion: Ara-C selection of MNs can be combined with stem cell differentiation protocols to enrich MNs in culture.
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