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Rotation project 5

Project leader:  

Dr. med. Marco Niesche
Assistenzarzt / Wissenschaftlicher Mitarbeiter
Klinik und Poliklinik für Neurochirurgie
Universitätsklinikum Carl Gustav Carus
der Technischen Universität Dresden
Fetscherstraße 74
01307 Dresden 

Project title:   Neuroepithelial 3D tubes as a tool for studying spinal cord regeneration after injury 
Involved SFB members (co-leaders):  

Prof. Dr. Elly Tanaka
für Regenerative Therapien Dresden (CRTD)
SFB project section A4

PD Dr. Matthias Kirsch
Klinik und Poliklinik für Neurochirurgie
Universitätsklinikum Carl Gustav Carus
der Technischen Universität Dresden

Prof. Dr. Gabriele Schackert
Klinik und Poliklinik für Neurochirurgie
Universitätsklinikum Carl Gustav Carus
der Technischen Universität Dresden

Funding period:   October 1, 2010 - September 30, 2011 

Objective: Traumatic spinal cord injury (SCI) is a devastating illness with severe disability, major associated morbidities and poor feasibility of regeneration. We know from regenerative amphibians, that reconstitution of SC structures is a highly ordered process that requires cellular dedifferentiation, proliferation and extension of an ependymal tube like structure. In this project we treat organotypic cysts from mouse ESC to accomplish patterning and spatial organization. Therefore we want to figure out appropriate conditions and techniques to pattern these neuroepithelial cysts.

Methods: Based on the model of creating 3D neuroepithelial cysts in vitro we use single mESC growing in a matrix. Cyst formation occurrs within few days under defined conditions. In order to shift them along the rostrocaudal axis and pattern in the dorsoventral orientation of developing SC, cysts are treated with several posteriorizing and dorsalizing i.e. ventralizing morphogens at distinct timepoints. Cyst formation, cellular development and differentiation processes were qualitatively analyzed and quantified using immunofluorescence, confocal microscopy, PCR and ISH.

Results: Under supplementary medium, embedded ESC grow clonally as cyst like structures. Treatment with RA caudalizes cysts to cervical levels of developing neural axis. Caudalized cysts can be shifted along the dorsoventral axis of developing SC. After incubation with shh-agonist ventral interneurons and motoneurons develop. Treatment with dorsalizing morphogens BMP and Wnt provides dorsal sensory cells. Fusing different types we generated polarized cysts with distinct dorso-ventral polarity indicating a spatial organization.

Conclusion: So far we developed a 3D cyst culture from mESC that recapitulates spatial and temporal neuroepithelial development and resembles the neural tube in vitro. Ependymal tube-like aggregates express embryonic antigens in defined spatial domains and react to locotypical morphogens with neuronal specification and patterning of neural tissue. Transplantable 3D aggregates should enhance regenerative ability of severed tissue, support axonal growth and restruction of the lesion. To examine the engraftment potential of the neuroepithelial cysts in vivo, in the near future we plan to implant the cysts in a rat SCI model to test tumorigenicity, axonal sprouting in various matrices, cellular and histological integration, functional improvement and maximum size of the SC lesion that can be restored. 

Relevant publications:  
  1. Cao QL, Zhang YP, Howard RM, Walters WM, Tsoulfas P, Whittemore SR: Pluripotentstem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. ExpNeurol 167:48-58, 2001
  2. Chow SY, Moul J, Tobias CA, et al.: Characterization and intraspinal grafting of EGF/bFGF-dependent neurospheres derived from embryonic rat spinal cord. Brain Res 874:87-106, 2000
  3. von EM, Janson AM, Larsen JO, Seiger A, Forno L, Bunge MB, Sundstrom E: pontaneous axonal regeneration in rodent spinal cord after ischemic injury. J Neuropathol Exp Neurol 61:64-75, 2002
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