gms | German Medical Science

60th Annual Meeting of the German Society of Neurosurgery (DGNC)
Joint Meeting with the Benelux countries and Bulgaria

German Society of Neurosurgery (DGNC)

24 - 27 May 2009, Münster

Embryonic stem cells release neurotrophins and differentiate along neural pathway after conditioning with brain extracts – „in-vitro Modelling of Stem Cell Behavior after Implantation following Traumatic Brain Injury“

Meeting Abstract

  • M. Molcanyi - Klinik und Poliklinik für Allgemeine Neurochirurgie, Klinikum der Universität zu Köln
  • K. Bentz - Institut für Entwicklungsgenetik, Helmholtz Zentrum, München
  • P. Riess - Lehrstuhl für Unfallchirurgie und Orthopädie, Köln-Merheim, Universität Witten-Herdecke
  • R. Ernestus - Klinik und Poliklinik für Allgemeine Neurochirurgie, Klinikum der Universität zu Köln
  • E. Neugebauer - Institut für Forschung in der Operativen Medizin (IFOM) in Köln-Merheim, Universität Witten-Herdecke
  • J. Hescheler - Neurophysiologie, Klinikum der Universität zu Köln
  • U. Schäfer - Universitätsklinik für Neurochirurgie, Graz, Österreich

Deutsche Gesellschaft für Neurochirurgie. 60. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), Joint Meeting mit den Benelux-Ländern und Bulgarien. Münster, 24.-27.05.2009. Düsseldorf: German Medical Science GMS Publishing House; 2009. DocMO.13-05

DOI: 10.3205/09dgnc093, URN: urn:nbn:de:0183-09dgnc0930

Published: May 20, 2009

© 2009 Molcanyi et al.
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Outline

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Objective: Transplantation of embryonic stem (ES) cells was shown to improve the neurologic outcome after traumatic brain injury in animal experiments. To clarify the underlying mechanisms, we established an in-vitro model depicting the behaviour of stem cells after implantation into traumatized brain.

Methods: Male Sprague-Dawley rats were subjected to a lateral fluid-percussion injury (TBI). 1 hr. after TBI, the ipsilateral cortex (n=20) was removed, homogenized and ultracentrifuged, until the supernatant was separated. The supernatant from healthy brains served as a control (n=10). Two stem cell lines - eGFP-transfected ES cells (D3-line) and CGR-8 ES cells were conditioned with a culture medium supplemented with 20% brain extract (supernatant) for a period of 7 days. The culture medium was changed on the 3rd, 5th and 7th day. ELISA was used to evaluate the production of neurotrophic factors in the ES cell cultures (NGF, BDNF an NT3). Real-time-PCR analysis (Oct4, Nestin, MAP2) as well as fluorescent immunocytochemistry were performed in order to investigate the differentiation status of cells conditioned by healthy vs. trauma-brain-extract.

Results: Conditioning of both ES cell lines with brain extracts (trauma and healthy) resulted in a significant time-dependent release of BDNF. The increase in BDNF release by the D-3 line was more pronounced when incubated with a trauma extract. The intrinsic capacity of the D-3 line to release BDNF and NT-3 exceeded the amount released by CGR8 cells by a factor of 10 and 4, respectively. NGF was secreted only by the D-3 line after stimulation with the trauma extract. Exposition to the healthy extract inhibited the release of both NGF (in the D-3 line) and NT-3 (in both lines). Both cell lines exposed to cerebral extracts differentiated along the neural pathway. Initially, both lines expressed an undifferentiated marker Oct-4. As soon as 3 days after first conditioning with healthy/trauma extracts, the moderate expression of early neural marker, Nestin, was observed in both cell lines. Further neural differentiation, indicated by MAP expression was seen in cells stimulated by the trauma extract. Immunocytochemistry, performed on conditioned ES cells, correlated with the above stated results and revealed specific neural phenotypes.

Conclusions: Authors observed a statistically significant production of neurotrophic factors and differentiation along neural pathway in investigated cell lines after conditioning with brain extracts. This phenomenon encourages further exploration of embryonic stem cells as possible therapeutics for traumatic brain injury. It also proves our model to be an useful in-vitro alternative, prior to implanting the ES cells in animal experiments.