Tyrosine Hydroxylase 유전자가 주입된 인간 배아줄기세포의 체외 신경세포 분화

In vitro Neural Cell Differentiation of Genetically Modified Human Embryonic Stem Cells Expressing Tyrosine Hydroxylase

  • 신현아 (마리아 기초의학연구소/마리아바이오텍) ;
  • 김은영 (마리아 기초의학연구소/마리아바이오텍) ;
  • 이금실 (마리아 기초의학연구소/마리아바이오텍) ;
  • 조황윤 (마리아 기초의학연구소/마리아바이오텍) ;
  • 김용식 (서울대학교 약리학교실) ;
  • 이원돈 (마리아불임병원) ;
  • 박세필 (마리아 기초의학연구소/마리아바이오텍) ;
  • 임진호 (마리아불임병원)
  • Shin, Hyun-Ah (Maria Infertility Hospital Medical Institute/Maria Biotech) ;
  • Kim, Eun-Young (Maria Infertility Hospital Medical Institute/Maria Biotech) ;
  • Lee, Keum-Sil (Maria Infertility Hospital Medical Institute/Maria Biotech) ;
  • Cho, Hwang-Yoon (Maria Infertility Hospital Medical Institute/Maria Biotech) ;
  • Kim, Yong-Sik (Seoul National University Dept. of Pharmacology) ;
  • Lee, Won-Don (Maria Infertility Hospital) ;
  • Park, Se-Pill (Maria Infertility Hospital Medical Institute/Maria Biotech) ;
  • Lim, Jin-Ho (Maria Infertility Hospital)
  • 발행 : 2004.03.30

초록

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 and Methods: 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 ($5{\times}10^{-2}mM$) 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 ($\sim$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.

키워드

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