title: A simple method for large-scale generation of dopamine neurons from human embryonic stem cells.
abstract: Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are potentially valuable in drug screening and as a possible source of donor tissue for transplantation in Parkinson's disease. However, existing culture protocols that promote the differentiation of DA neurons from hESCs are complex, involving multiple steps and having unreliable results between cultures. Here we report a simple and highly reproducible culture protocol that induces expandable DA neuron progenitors from hESCs in attached cultures. We found that the hESC-derived neuronal progenitors retain their full capacity to generate DA neurons after repeated passaging in the presence of basic fibroblast growth factor (bFGF) and medium conditioned with PA6 stromal cells. Using immunocytochemistry and RT-PCR, we found that the differentiated DA neurons exhibit a midbrain phenotype and express, e.g., Aldh1a, Ptx3, Nurr1, and Lmx1a. Using HPLC, we monitored their production of DA. We then demonstrated that the expanded progenitors are possible to cryopreserve without loosing the dopaminergic phenotype. With our protocol, we obtained large and homogeneous populations of dopaminergic progenitors and neurons. We conclude that our protocol can be used to generate human DA neurons suitable for the study of disease mechanisms, toxicology, drug screening, and intracerebral transplantation.
authors.affiliation: Neuronal Survival Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden. asuka.morizane@frontier.kyoto-ac.jp
authors.forename: Asuka
authors.initials: A
authors.lastname: Morizane
authors.forename: Vladimer
authors.initials: V
authors.lastname: Darsalia
authors.forename: M Oktar
authors.initials: MO
authors.lastname: Guloglu
authors.forename: Tord
authors.initials: T
authors.lastname: Hjalt
authors.forename: Manolo
authors.initials: M
authors.lastname: Carta
authors.forename: Jia-Yi
authors.initials: JY
authors.lastname: Li
authors.forename: Patrik
authors.initials: P
authors.lastname: Brundin
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D018920:Coculture Techniques / Q000379:methods; D004298:Dopamine / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D055495:Neurogenesis / Q000502:physiology*; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D017154:Stromal Cells / Q000166:cytology
chemical_list: D004298:Dopamine