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Korean Journal of Fertility and Sterility 2004;31(1):19-27.
Published online March 1, 2004.
Effects of Neurotrophic Factors on the Generation of Functional Dopamine Secretory Neurons Derived from in vitro Differentiated Human Embryonic Stem Cells.
Keum Sil Lee, Eun Young Kim, Hyun Ah Shin, Hwang Yoon Cho, Kyu Chang Wang, Yong Sik Kim, Hoon Taek Lee, Kil Saeng Chung, Won Don Lee, Sepill Park, Jin Ho Lim
1Maria Infertility Hospital Medical Institute/Maria Biotech, Korea.
2Seoul National University Department of Neurosurgery, Korea.
3Seoul National University Department of Pharmacology, Korea.
4Konkuk University, Korea.
5Maria Infertility Hospital, Korea.
Abstract
OBJECTIVE
This study was to examine the in vitro neural cell differentiation patterns of human embryonic stem (hES) cells following treatment of various neurotrophic factors [basic fibroblast growth factor (bFGF), retinoic acid (RA), brain derived neurotrophic factor (BDNF) and transforming growth factor (TGF)-alpha], particulary in dopaminergic neuron formation. METHODS: The hES cells were induced to differentiate by bFGF and RA. Group I) In bFGF induction method, embryoid bodies (EBs, for 4 days) derived from hES were plated onto gelatin dish, selected for 8 days in ITSFn medium and expanded at the presence of bFGF (10 ng/ml) for another 6 days followed by a final differentiation in N2 medium for 7, 14 and 21 days. Group II) For RA induction, EBs were exposed of RA (10-6 M) for 4 days and allowed to differentiate in N2 medium for 7, 14 and 21 days. Group III) To examine the effects of additional neurotrophic factors, bFGF or RA induced cells were exposed to either BDNF (10 ng/ml) or TGF-alpha (10 ng/ml) during the 21 days of final differentiation. Neuron differentiation and dopamine secretion were examined by indirect immunocytochemistry and HPLC, respectively. RESULTS: The bFGF or RA treated hES cells were resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with BDNF or TGF-alpha during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression of a dopaminergic neuron marker, compared to control (p<0.05). In contrast, no effect was observed on the rate of mature neuron (NF-200) or glutamic acid decarboxylase-positive neurons. Immunocytochemistry and HPLC analyses revealed the higher levels of TH expression (20.3%) and dopamine secretion (265.5+/-62.8 pmol/mg) in bFGF and TGF-alpha sequentially treated hES cells than those in RA or BDNF treated hES cells. CONCLUSION: These results indicate that the generation of dopamine secretory neurons from in vitro differentiated hES cells can be improved by TGF-alpha addition in the bFGF induction protocol.
Key Words: Human embryonic stem cell; Neural cell differentiation; Basic fibroblast growth factor; Transforming growth factor-alpha; Dopaminergic neuron
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