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Efficient Diff of mES into DA neurons

Efficient Diff of mES into DA neurons - DEVELOPMENTAL...

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Rapid and e⁄cient di¡erentiation of dopaminergic neurons from mouse embryonic stem cells Thorsten Lau, Sylvia Adam and Patrick Schloss Biochemical Laboratory,Central Institute of Mental Health, Mannheim,Germany Correspondence and requests for reprints to Dr. Patrick Schloss, Biochemical Laboratory,Central Institute of Mental Health, 68159 Mannheim,Germany Tel: 49 6211703 2901; fax: 49 6211703 6252; e-mail: [email protected] Sponsorship:This work was supported by the Deutsche Forschungsgemeinschaft (SFB636). Received 3 April 2006; accepted11April 2006 We have developed a fast and e¡ective method for the di¡erentia- tion of dopaminergic neurons from mouse embryonic stem cells. Neuronal precursors are obtained by formation of embryonic bodies or neural stem spheres via free-£oating culture in the pre- sence of the mitogensbasic ¢broblastgrowth factor and epidermal growth factor together with L -ascorbic acid. Subsequent culturing of the precursor cells in medium containing epidermal growth factor, FGF8b, SHH and ascorbic acid induces cell proliferation, following withdrawal of the growth factors leads di¡erentiation into predominantly dopaminergic neurons. Mature neurons are obtained within10 days of replacing the proliferation to di¡erentia- tion medium. Embryonic stem-derived dopaminergic neurons are puri¢ed by cell sorting and may serve as a convenient source for the study of molecular, genetic and cellular properties of dopa- minergic neurons. NeuroReport 17:975^979 ± c 2006 Lippincott Williams & Wilkins. Keywords : embryonic stem cells, dopaminergic neurons, neuronal di¡erentiation Introduction Mouse embryonic stem (ES) cells are derived from the inner cell mass of growing blastocysts and are considered to be pluripotent as they are able to proliferate and differentiate into a variety of endodermal, mesodermal and ectodermal cell types, including neurons [1–3]. These features of ES cells are of special interest in neuroscience research because they can serve as a source for homogenous and large neuronal cell populations for biochemical approaches. Several proto- cols have been described so far for the differentiation of dopaminergic neurons from ES cells via the formation of embryoid bodies (EBs) [4–7] or stromal cell-derived- inducing activity [8,9]. These protocols, however, prove to be rather time consuming. In this study, we have established new methods for the efficient differentiation of dopaminergic neurons from mouse ES cells. These methods are based on the formation of either EBs or neuronal stem spheres (NSS) by free-floating culture and take only about 14 days to produce predominantly dopaminergic neurons. Essentially, we have shortened the time for the generation of neuronal precursors by adding defined growth factors already during EB and NSS formation, respectively. In addition, we have supplemented the medium with L -ascorbic acid, and replaced basic fibroblast growth factor (bFGF) with fibroblast growth factor (FGF) 8b and sonic hedgehog (SHH) during cell proliferation which enhances the dopaminergic fate of neurons during differentiation.
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