gms | German Medical Science

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 are a highly ordered process that requires cellular dedifferentiation, proliferation and extension of an ependymal tube like structure. In this project we wanted to treat organotypic cysts from mouse ESC to accomplish patterning and spatial organization. Therefore we wanted to figure out appropriate conditions and techniques to pattern these neuroepithelial cysts.

Method: Based on the model of creating 3D neuroepithelial cysts in vitro we used single mESC growing in a matrix. Cyst formation occurred 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 were 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.

Conclusions: We have developed a 3D mouse neuroepithelial cyst culture starting from mouse embryonic stem cells (mESCs) that remarkably recapitulates the spatial organization of neuroepithelium, and the tempo of neural development. The ependymal tube-like cellular aggregates from organotypic cultures of murine ES cells display neuroepithelial differentiation pattern and resemble the neural tube in vitro. Such transplantable 3D aggregates should be used to study formation, growth and differentiation of the embryonic temporal and spatial coordinate system in the developing CNS as well as a tool for enhancing the regenerative ability of severed CNS tissue to support restruction of the lesion after transplantation of single cysts directly into the injured site.