In vitro Neural Cell Differentiation of Genetically Modified Human Embryonic Stem Cells Expressing Tyrosine Hydroxylase / 대한불임학회지
Korean Journal of Fertility and Sterility
;
: 67-74, 2004.
Article
in Korean
| WPRIM
| ID: wpr-64691
ABSTRACT
OBJECTIVE:
This study was to examine in vitro neural cell differentiation pattern of the genetically modified human embryonic stem cells expressing tyrosine hydroxylase (TH). MATERIALS ANDMETHODS:
Human embryonic stem (hES, MB03) cell was transfected with cDNAs cording for TH. Successful transfection was confirmed by western immunoblotting. Newly transfected cell line (TH#2/MB03) was induced to differentiate by two neurogenic factors retinoic acid (RA) and b-FGF. Exp. I) Upon differentiation using RA, embryoid bodies (EB, for 4 days) derived from TH#2/MB03 cells were exposed to RA (10-6 M)/AA (5x10-2 mM) for 4 days, and were allowed to differentiate in N2 medium for 7, 14 or 21 days. Exp. II) When b-FGF was used, neuronal precursor cells were expanded at the presence of b-FGF (10 ng/ml) for 6 days followed by a final differentiation in N2 medium for 7, 14 or 21 days. Neuron differentiation was examined by indirect immunocytochemistry using neuron markers (NF160 & NF200).RESULTS:
After 7 days in N2 medium, approximately 80% and 20% of the RA or b-FGF induced Th#2/MB03 cells were immunoreactive to anti-NF160 and anti-NF200 antibodies, respectively. As differentiation continued, NF200 in RA treated cells significantly increased to 73.0% on 14 days compared to that in b-FGF treated cells (53.0%, p<0.05), while the proportion of cells expressing NF160 was similarly decreased between two groups. However, throughout the differentiation, expression of TH was maintained (~90%). HPLC analyses indicated the increased levels of L-DOPA in RA treated genetically modified hES cells with longer differentiation time.CONCLUSION:
These results suggested that a genetically modified hES cells (TH#2/MB03) could be efficiently differentiated in vitro into mature neurons by RA induction method.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Tyrosine
/
Tretinoin
/
Tyrosine 3-Monooxygenase
/
Immunohistochemistry
/
Transfection
/
Levodopa
/
Cell Differentiation
/
Cell Line
/
Blotting, Western
/
Chromatography, High Pressure Liquid
Limits:
Humans
Language:
Korean
Journal:
Korean Journal of Fertility and Sterility
Year:
2004
Type:
Article
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