gms | German Medical Science

Objective: Affection of CNS tissue accompanies with devastating morbidities and only limited potential for recovery. So far, different approaches replenishing the cellular pool in the defects had only limited success. This study aims to generate different progenitor cell types of the developing CNS in a 3D organotypic pattern from murine embryonic stem cells (mESC). Formation of neuroepithelial tube-like structures in vitro could serve as a source for tissue reconstitution in lesioned CNS.

Method: In the present study a 3D cell culture system for neural progenitors was used, to reproduce development of the embryonic CNS. Neuroepithelial tubes were generated from single mESC in a 3D matrix under neurosupplementary medium. They were differentiated into cellular subtypes subsequently by locotypic morphogens and growth factors (GF). Cellular development and differentiation was qualitatively analyzed and quantified using immunofluorescence, confocal microscopy, PCR and ISH.

Results: Matrigel embedded mESC grow clonally in N2B27 medium as tubes and follow embryonic development. They possess apical-basal polarity, express neural stem cell markers and differentiate along the neural lineage. IF and ISH data suggest a midbrain-hindbrain localization of mature tubes. They can be shifted along the craniocaudal and dorsoventral axis of developing SC. RA treatment causes caudalization. Shh induces formation of ventral, whereas BMP and Wnt proteins generate dorsal neurons. Growth factor treatment with EGF and FGF provides upregulation of BLBP and RC2 up to 80% of treated cells, indicating radial glia development.

Conclusions: 3D neuroepithelial tube culture remarkably recapitulates the spatial organization of a developing CNS. Ependymal cellular aggregates react to locotypical morphogens with specification and resemble the neural tube in vitro. Radial glia cells as a typical source of CNS regeneration were created in high efficiency, in order to support restruction of lesioned CNS and to aggravate neuroregenenerative ability. Engraftment potential, cellular and histological integration, tumorigenicity and functional improvement of those cells are still to be examined in a transplantation essay.