Journal
PLOS ONE
Volume 10, Issue 9, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0138460
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Funding
- Medical Research Center through the National Research Foundation of Korea (NRF) - the Ministry of Science, ICT & Future Planning, Hanyang University [2008-0062190]
- Basic Science Research Program [2011-0008952]
- Bio & Medical Technology Development Program through the National Research Foundation of Korea (NRF) - Ministry of Science, ICT & Future Planning, Hanyang University [201400000000578]
- Rural Development Administration, Republic of Korea [PJ008022032012]
- National Research Foundation of Korea [2008-0062190] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
- Rural Development Administration (RDA), Republic of Korea [PJ008022032012] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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Directed methods for differentiating human embryonic stem cells (hESCs) into dopaminergic (DA) precursor cells using stromal cells co-culture systems are already well established. However, not all of the hESCs differentiate into DA precursors using these methods. HSF6, H1, H7, and H9 cells differentiate well into DA precursors, but CHA13 and CHA15 cells hardly differentiate. To overcome this problem, we modified the differentiation system to include a co-culturing step that exposes the cells to noggin early in the differentiation process. This was done using.-irradiated noggin-overexpressing CF1-mouse embryonic fibroblasts (MEF-noggin) and MS5 stromal cells (MS5-noggin and MS5-sonic hedgehog). After directed differentiation, RT-PCR analyses revealed that engrailed-1 (En-1), Lmx1b, and Nurr1, which are midbrain DA markers, were expressed regardless of differentiation stage. Moreover, tyrosine hydroxylase (Th) and an A9 midbrain-specific DA marker (Girk2) were expressed during differentiation, whereas levels of Oct3/4, an undifferentiated marker, decreased. Immunocytochemical analyses revealed that protein levels of the neuronal markers TH and TuJ1 increased during the final differentiation stage. These results demonstrate that early noggin exposure may play a specific role in the directed differentiation of DA cells from human embryonic stem cells.
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