Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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In Vitro Expansion of Homogeneous Neural Precursor Cells Derived from Human Embryonic Stem Cells

  • Na, Deuk-Chae (CHA Stem Cell Institute, Pochon CHA University College of Medicine) ;
  • Kim, Se-Hee (CHA Stem Cell Institute, Pochon CHA University College of Medicine) ;
  • Choi, Won-Ik (CHA Stem Cell Institute, Pochon CHA University College of Medicine) ;
  • Hwang, Hyun-Jin (CHA Stem Cell Institute, Pochon CHA University College of Medicine) ;
  • Han, In-Bo (Department of Neurosurgery Bundang CHA Hospital) ;
  • Kim, Jae-Hwan (CHA Stem Cell Institute, Pochon CHA University College of Medicine) ;
  • Park, Keun-Hong (CHA Stem Cell Institute, Pochon CHA University College of Medicine) ;
  • Chung, Hyung-Min (CHA Stem Cell Institute, Pochon CHA University College of Medicine) ;
  • Choi, Seong-Jun (CHA Stem Cell Institute, Pochon CHA University College of Medicine)
  • Published : 2007.12.31
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Abstract

Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and have the capacity to differentiate into various types of cells in the body. Hence, these cells may potentially be an indefinite source of cells for cell therapy in various degenerative diseases including neuronal disorders. For clinical applications of human ES cells, directed differentiation of these cells would be necessary. The objective of this study is to develop the culture condition for the expansion of neural precursor cells derived from human ES cells. Human ES cells were able to differentiate into neural precursor cells upon a stepwise culture condition. Neural precursor cells were propagated up to 5000-fold in cell numbers over 12-week period of culture and evaluated for their characteristics. Expressions of sox1 and pax6 transcripts were dramatically up-regulated along the differentiation stages by RT-PCR analysis. In contrast, expressions of oct4 and nanog transcripts were completely disappeared in neural precursor cells. Expressions of nestin, pax6 and sox1 were also confirmed in neural precursor cells by immunocytochemical analysis. Upon differentiation, the expanded neural precursor cells differentiated into neurons, astrocytes, and oligodendrocytes. In immunocytochemical analysis, expressions of type III ${\beta}$-tubulin and MAP2ab were observed Presence of astrocytes and oligodendrocytes were also confirmed by expressions of GFAP and O4, respectively. Results of this study demonstrate the feasibility of long-term expansion of human ES cell-derived neural precursor cells in vitro, which can be a potential source of the cells for the treatment of neurodegenerative disorders.

Keywords

References

  1. Joannides AJ, Fiore-Heriche C, Battersby AA, Athauda-Arachchi P, Bouhon lA, Williams L, Weshnore K, Kemp PJ, Compston A, Allen ND, Chandran S (2007): A scaleable and defined system for generating neural stem cells from human embryonic stem cells. Stem Cells 25:731-737 https://doi.org/10.1634/stemcells.2006-0562
  2. Campos LS (2004): Neurospheres: insights into neural stem cell biology. J Neurosci Res 78:761-769 https://doi.org/10.1002/jnr.20333
  3. Carpenter MK, Inokuma MS, Denham J, Mujtaba T, Chiu CP, Rao MS (2001): Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 172:383-397 https://doi.org/10.1006/exnr.2001.7832
  4. Conover JC, Ip NY, Poueymirou WT, Bates B, Goldfarb MP, DeChiara 1M, Yancopoulos GD (1993): Ciliary neurotrophic factor maintains the pluripotentiality of embryonic stem cells. Development 119: 559-565
  5. Dunnett SB, Bjorklund A (1999): Prospects for new restorative and neuroprotective treatments in Parkinson's disease. Nature 399:A32-39 https://doi.org/10.1038/19899
  6. Grilli A, Cova L, Parati EA, Galli R, Vescovi AL (1995): Basic fibroblast growth factor supports the proliferation of epidermal growth factor-generated neuronal precursor cells of the adult mouse CNS. Neurosci Lett 185:151-154 https://doi.org/10.1016/0304-3940(95)11247-T
  7. Hagell P, Schrag A, Piccini P, Jahanshahi M, Brown R, Rehncrona S, Widner H, Brundin P, Rothwell JC; Odin P, Wenning GK, Morrish P, Gustavii B, Bjorklund A, Brooks DJ, Marsden CD, Quinn NP, Lindvall O (1999): Sequential bilateral transplantation in Parkinson's disease: effects of the second graft. Brain 122:1121-1132 https://doi.org/10.1093/brain/122.6.1121
  8. Hoffer BJ, Leenders KL, Young D, Gerhardt G, Zerbe GO, Bygdeman M, Seiger A, Olson L, Stromberg I, Freedman R (1992): Eighteen-month course of two patients with grafts of fetal dopamine neurons for severe Parkinson's disease. Exp Neurol 118: 243-252 https://doi.org/10.1016/0014-4886(92)90181-O
  9. Ip NY, McOain J, Barrezueta NX, Aldrich TH, Pan L, Li Y, Wiegand SJ, Friedman B, Davis S, Yancopoulos GD (1993): The alpha component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development. Neuron 10:89-102 https://doi.org/10.1016/0896-6273(93)90245-M
  10. Ko J-Y, Park C-H, Koh H-C, Cho Y-H, Kyhm J-H, Kim Y-S, Lee I, Lee Y-S, Lee S-H (2007): Human embryonic stem cell-derived neural precursors as a continuous, stable, and on-demand source for human dopamine neurons. J Neurochem. 10:1471-4159
  11. Kalyani A, Hobson K, Rao MS (1997): Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterization, and clonal analysis. Dev Biol 186:202-223 https://doi.org/10.1006/dbio.1997.8592
  12. Nunes MC, Roy NS, Keyoung HM, Goodman RR, McKhann G, Jiang L, Kang J, Nedergaard M, Goldman SA (2003): Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat Med 9:439-447 https://doi.org/10.1038/nm837
  13. Olanow CW (1992): Early therapy for Parkinson's disease. Eur Neurol 32 Suppl 1:30-35 https://doi.org/10.1159/000116867
  14. Palmer TD, Takahashi J, Gage FH (1997): The adu1t rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 8:389-404 https://doi.org/10.1006/mcne.1996.0595
  15. Reynolds BA, Tetzlaff W, Weiss S (1992): A multipotent EGF-responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12: 4565-4574 https://doi.org/10.1523/JNEUROSCI.12-11-04565.1992
  16. Reynolds BA, Weiss S (1996): Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 175:1-13 https://doi.org/10.1006/dbio.1996.0090
  17. Chung S, Shin B-S, Hwang M, Lardaro T, Kang UJ, Isacson O, Kim K-S (2006): Neural precursors derived from embryonic stem cells, but not those from fetal ventral mesencephalon, maintain the potential to differentiate into dopaminergic neurons after expansion in vitro. Stem Cells 24:1583-1593 https://doi.org/10.1634/stemcells.2005-0558
  18. Kim S, Hong JY, Joo SY, Kim JH, Moon SY, Yoon HS, Kim DH, Chung HM, Choi S-J (2004): Derivation of neural precursor cells from human embryonic stem cells. Reprod Dev Biol 28:247-252
  19. Shin S, Mitalipova M, Noggle S, Tibbitts D, Rao A VR, Stice SL (2006): Long-term proliferation of human embryonic stem cell- derived neuroepithelial cells using defined adherent culture conditions. Stem Cells 24:125-138 https://doi.org/10.1634/stemcells.2004-0150
  20. Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, van der Kooy D (1999): Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 208:166-188 https://doi.org/10.1006/dbio.1998.9192
  21. Ying QL, Stavridis M, Griffiths D, Li M, Smith A (2003): Conversion of embryonic stem cells into neuroectodermal precursors in adherent monocu1ture. Nat Biotechnol 21:183- 186 https://doi.org/10.1038/nbt780
  22. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001): In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19:1129-1133 https://doi.org/10.1038/nbt1201-1129