期刊
JOURNAL OF NEUROSCIENCE RESEARCH
卷 88, 期 16, 页码 3467-3478出版社
WILEY-BLACKWELL
DOI: 10.1002/jnr.22515
关键词
drug screening; Parkinson's disease; transplantation; neural progenitors; pluripotent stem cells
资金
- Swedish Research Council
- Swedish Parkinson Foundation
- Brain Foundation
- Soderberg Foundation
- Olle Engqvist Byggmastare Foundation
- Marie Curie Initial Training Network
- Grants-in-Aid for Scientific Research [21591837] Funding Source: KAKEN
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. (C) 2010 Wiley-Liss, Inc.
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