gms | German Medical Science

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2017)

24.10. - 27.10.2017, Berlin

Autologous multipotent stem cells and ensheathing cells from human olfactory mucosa for neuroregenerative approaches

Meeting Abstract

  • presenting/speaker Christina Sengstock - BG Universitätsklinikum Bergmannsheil, Chirurgische Forschung, Bochum, Germany
  • Nora Knips - BG Universitätsklinikum Bergmannsheil, Chirurgische Forschung, Bochum, Germany
  • Amir Minovi - St. Elisabeth Hospital, Katholisches Krankenhaus, Klinik für Hals-, Nasen- und Ohrenheilkunde, Ruhr-Universität Bochum, Bochum, Germany
  • Thomas A. Schildhauer - BG Universitätsklinikum Bergmannsheil, Chirurgische Forschung, Bochum, Germany
  • Stefan Dazert - St. Elisabeth Hospital, Katholisches Krankenhaus, Klinik für Hals-, Nasen- und Ohrenheilkunde, Ruhr-Universität Bochum, Bochum, Germany
  • Manfred Köller - Ruhr-Universität Bochum, BG Universitätsklinikum Bergmannsheil, Chirurgische Forschung, Bochum, Germany

Deutscher Kongress für Orthopädie und Unfallchirurgie (DKOU 2017). Berlin, 24.-27.10.2017. Düsseldorf: German Medical Science GMS Publishing House; 2017. DocPO12-580

doi: 10.3205/17dkou615, urn:nbn:de:0183-17dkou6152

Veröffentlicht: 23. Oktober 2017

© 2017 Sengstock et al.
Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). Lizenz-Angaben siehe http://creativecommons.org/licenses/by/4.0/.


Gliederung

Text

Objectives: One promising strategy to improve the neurological function after traumatic spinal cord injuries (SCI) could be an autologous cell therapy. Due to the lifelong neurogenesis the olfactory mucosa might be an attractive cell source. The neuro-regenerative properties are attributed to the olfactory multipotent stem cells (OSC) and to the olfactory ensheathing cells. The regenerative capacity of these cells has already been successfully demonstrated in animal models and some case reports. Up to now, there are no precise and standard methods for cell isolation and characterization. In this study we described a method to isolate OSC from human olfactory mucosa and characterize the isolated cells in comparison to bone marrow mesenchymal stem cells. In addition, the viability of these isolated cells were analyzed in an autologous plasma clot matrix as a possible carrier matrix for cell transplantation.

Methods: Human olfactory mucosa biopsies were collected from 4 individuals during corrections of the nasal septum and cut into pieces of 200 - 500 µm. After outgrowing of cells, adherent cells were expanded in cell culture flasks. For assay of neurosphere formation, cells were plated at 5,000 - 10,000 cells/cm2 in DMEM/ FCS. After adhesion of the cells, medium was changed to serum-free DMEM + 50 ng/ml EGF (epidermal growth factor) + 50 ng/ml FGF (fibroblast growth factor) + 1% ITS (human insulin, transferrin, sodium selenite). After neurospheres had formed, the wells were gently washed to collect floating neurospheres. Typical surface markers such as CD90, CD105, p75NTR, CD44 and nestin were analyzed by using the flow cytometry and differentiation capacity into neuronal lineage (MAP-2 expression) was analyzed by using confocal laser scanning microscopy. To verify the osteogenic differentiation potential, cells were incubated with an osteogenic differentiation kit for 21 days and differentiation was measured by Alizarin Red S staining. Human bone marrow-derived mesenchymal stem cells (hMSC) were used for comparison. Plasma clots (citrated blood, centrifugation 2000 x g, 45 min) were prepared by adding plasma and OSC in a seeding density of 104 cells. Viability within the plasma clot was analyzed by calcein-AM/ propidiumiodide staining.

Results and Conclusion: We demonstrated that OSC can be isolated from the olfactory mucosa and are closely related to bone hMSC. Human olfactory cells proliferate faster than hMSC and express typical hMSC surface markers such as CD90 or CD105 and show a lack of expression of CD45 and CD31. Expanded cells are able to form neurospheres and differentiate into neuron-like cells expressing MAP-2. In addition, the isolated neurosphere-forming cells can differentiate into osteogenic lineage and express typical markers such as p75NTR and GFAP and are positive for nestin. We have shown that olfactory cells stay viable and are able to proliferate within a three-dimensional plasma clot, which might be a favourable matrix for cell transplantation at spinal cord sites.