Engineering of Three Dimensional Micro Nerve Tissue Using Postnatal Stem Cells from Human Dental Apical Papilla

The generation of cell-based three dimensional (3D) nerve tissue is an attractive subject to improve graft survival and integration into host tissue for neural tissue regeneration or to model biological events in stem cell differentiation. However, although 3D organotypic culture strategy has been well established for 3D nerve tissue formation of pluripotent stem cells to study underlying biology in nerve development, cell-based nerve tissue have not been developed using human postnatal stem cells with therapeutic potential of stem cell-based therapy. Here, we established culture strategy for the generation of in vitro cell-based 3D nerve tissue from postnatal stem cells from apical papilla (SCAPs) of tooth, which is known to be originated from neural crest-derived ectomesenchyme. A stem cell population capable of differentiating into neural cell lineages was generated during the ex-vivo expansion of SCAPs in the presence of EGF and bFGF, and SCAPs differentiated into neural cells showing neural cell lineage-related molecular and gene expression profiles with morphological change under neural inductive culture condition. Moreover, we showed the first evidence that 3D cell-based nerve-like tissue with axon and myelin structure could be generated from SCAPs via 3D organotypic culture using an integrated bioprocess composing of polyethylene glycol (PEG) microwell-mediated cell spheroid formation and subsequent dynamic culture in a high aspect ratio vessel (HARV) bioreactor. In conclusion, our study propose that the culture strategy provide a novel approach to develop in vitro engineered nerve tissue using SCAPs and a foundation to study biological events in neural differentiation of postnatal stem cells. For DNA microarray analysis of the ex-vivo expanded stem cells derived from apical papillas, RNA was extracted from the ex-vivo expanded SCAPs at passage of 3, 6 and 9.