Identi fication of neurospheres generated from human dental pulp stem cells in xeno-/serum-free conditions

Introduction: Cell-based therapies require an emerging alternative treatment using easily harvested cell sources. Neural stem cells derived from various tissues, including brain, bone marrow, skin and retina can give rise to both neurons and glial cells. Recently, human dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to have mesenchymal stem cell-like abilities such as self-renewal and multi-lineage differentiation, including neuron and glial cells. Moreover, DPSCs and SHED show a higher proliferation rate and a higher number of population doublings compared with adult bone marrow stromal stem cells. Therefore, DPSCs are a useful source that can be applied in cell replacement therapy for various neurological disorders. Generally, the conventional culture methods for DPSCs have used serum, therefore the unde fined components in culture medium may complicate investigations of the molecular mechanisms that control the self-renewal and differentiation of DPSCs. However, neural stem cells proliferate to form neurospheres in suspension in vitro in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). No study to date has obtained neurospheres from DPSCs in serum-free conditions in primary culture. Thus, the aim of this study was to establish a method for the proliferation and neural differentiation of DPSCs in xenoand serum-free conditions in primary culture. Methods: DPSCs were obtained from the dental pulp of wisdom teeth from healthy individuals (18-41 years old) and cultured in conventional medium containing 15% fetal bovine serum and xeno-/serumfree medium. We evaluated the proliferation of DPSCs, neurosphere generation, and neural differentiation under xeno-/serum-free conditions by flow cytometry, immunohistochemistry, and real-time polymerase chain reaction. Results: In proliferation medium without xeno/serum, DPSCs can proliferate and generate neurospheres, however, the neurospheres had limited self-renewal ability. Under differentiation conditions, class III beta-tubulin (TUBB3) and microtubule-associated protein (MAP2) were more signi ficantly expressed in neurospheres derived from DPSCs in xeno-/serum-free culture conditions than in DPSCs in conventional culture conditions. Conclusions: Our result demonstrated that neurosphere generation from DPSCs in xeno-/serum-free culture may be an accessible source for clinical cell replacement therapies for neuronal degenerative diseases.