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

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

References

  1. Isacson O. The production and use of cells as therapeutic agents in neurodegenerative diseases. Lancet Neurol. 2003; 2(7): 417-24 https://doi.org/10.1016/S1474-4422(03)00437-X
  2. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of trans-plantable neural precursors from human embryonic stem cells. Nat Biotech 2001; 19: 1129-33 https://doi.org/10.1038/nbt1201-1129
  3. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T. Neural pro-genitors from human embryonic stem cells. Nat Biotech 2001; 19: 1134-40 https://doi.org/10.1038/nbt1201-1134
  4. Kim JH, Auerbach JM, Rodrigues-Gomez JA, Velasco I, Gavi D, Lumesky N, et al. Dopa-mine neurons derived from embryonic stem cells function in an animal model of Park-inson's disease. Nature 2002; 418: 50-6 https://doi.org/10.1038/nature00900
  5. Lee SH, Lumelsky N, Studer L, Auerbach JM, Mckay RD. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotech 2000; 18: 675-9 https://doi.org/10.1038/76536
  6. Rolletschek A, Chang H, Guan K, Czyz J,Meyer M, Wobus AM. Differentiation of embryo-nic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors. Mech Dev 2001; 105: 93-104 https://doi.org/10.1016/S0925-4773(01)00385-9
  7. Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton DA, Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 2000; 97: 11307-12 https://doi.org/10.1073/pnas.97.21.11307
  8. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science 1998; 282: 1145-7 https://doi.org/10.1126/science.282.5391.1145
  9. Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu CP, Rao MS. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurology 2001; 172: 383-97 https://doi.org/10.1006/exnr.2001.7832
  10. Schuldiner M, Eiges R, Eden A, Yanuka O, Itskovitz-Eldor J, Goldstein RS, et al. Induced neuronal differentiation of human embryonic stem cells. Brain Research 2001; 913: 201-5 https://doi.org/10.1016/S0006-8993(01)02776-7
  11. NIH Human Embryonic Stem Cell Registry. Na-tional Institutes of Health. USA. http://stemcells.nih.gov/registry/ eligibilityCriteria.asp
  12. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G. Structural characterization and bio-logical functions of fibroblast growth factor. Endocr Rev. 1987; 8(2): 95-114 https://doi.org/10.1210/edrv-8-2-95
  13. Bain G, Kitchens D, Yao M, Huettner JE, Gottlieb DI. Embryonic stem cells express neu ronal properties in vitro. Dev Biol 1995; 168: 342-57 https://doi.org/10.1006/dbio.1995.1085
  14. Maltsev VA, Rohwedel J, Hescheler J, Wobus AM. Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusodal, atrial and ventricular cell types. Mech Dev 1993; 44: 41-50 https://doi.org/10.1016/0925-4773(93)90015-P
  15. Rohwedel J, Maltsev V, Bober E, Arnold HH, Hescheler J, Wobus AM. Muscle cell differen tiation of embryonic stem cells reflects myo-genesis in vivo: developmentally regulated expression of myogenic determination genes and functional expression of ionic currents. Dev Biol 1994; 164: 87-101 https://doi.org/10.1006/dbio.1994.1182
  16. Dani C, Smith AG, Dessolin S, Leroy P, Staccini L, Villageois P, Darimont C, Ailhaud G, Differ-entiation of embryonic stem cells into adipocytes in vitro. J Cell Sci 1997; 110: 1279-85
  17. Drab M, Haller H, Bychkov R, Erdmann B, Lindschau C, Haase,H, et al. From totipotent embryonic stem cells to spontaneously contrac-ting smooth muscle cells a retinoic acid and db-cAMP in vitro differentiation model. FASEB J 1997; 11: 905-15 https://doi.org/10.1096/fasebj.11.11.9285489
  18. Ahmed S, Reynolds BA, Weiss S. BDNF en-hances the differentiation but not the survival of CNS stem cell-derived neuronal precursors. J Neurosci 1995; 15: 5765-78
  19. Murphy DD, Cole NB, Segal M. Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neu-rons. Proc Natl Acad Sci USA 1998; 95: 11412-7 https://doi.org/10.1073/pnas.95.19.11412
  20. Schreiber AB, Winkler MW, Derynck R. Trans-forming growth factor-a: a more potent angio-genic mediator than epidermal growth factor. Science 1986; 232: 1250-3 https://doi.org/10.1126/science.2422759
  21. Svendsen CN, Smith AG. New prospects for human stem-cell therapy in the nervous system. Trends Neurosci 1999; 22: 357-64 https://doi.org/10.1016/S0166-2236(99)01428-9