Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
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Effects of Neurotrophic Factors on the Generation of Functional Dopamine Secretory Neurons Derived from in vitro Differentiated Human Embryonic Stem Cells

신경성장촉진 인자가 인간 배아줄기세포 유래 도파민 분비 신경세포형성에 미치는 영향

  • 이금실 (마리아 기초의학연구소/마리아바이오텍) ;
  • 김은영 (마리아 기초의학연구소/마리아바이오텍) ;
  • 신현아 (마리아 기초의학연구소/마리아바이오텍) ;
  • 조황윤 (마리아 기초의학연구소/마리아바이오텍) ;
  • 왕규창 (서울대학교 신경외과학교실) ;
  • 김용식 (서울대학교 약리학교실) ;
  • 이훈택 (건국대학교) ;
  • 정길생 (건국대학교) ;
  • 이원돈 (마리아불임병원) ;
  • 박세필 (마리아 기초의학연구소/마리아바이오텍) ;
  • 임진호 (마리아불임병원)
  • Published : 2004.03.30
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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 $\pm$ 62.8 pmol/mg) in bFGF and TGF-sequentially treated hES cells than those in $\alpha$ 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.

Keywords

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