Highly Efficient Neural Differentiation of Human Somatic Stem Cells, Isolated by Minimally Invasive Periodontal Surgery

Abstract

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

A two-step strategy for neuronal differentiation in vitro of human dental follicle cells

“…However, less was known about neural differentiation potential of DFCs (Kemoun et al, 2007;Morsczeck et al, 2005a, b). In our study naïve DFCs strongly expressed early neural cell markers and on a low level markers for mature neural cells Corti et al, 2003;Wachs et al, 2003;Widera et al, 2007). The expression of cell markers can be identified in naïve DFCs by both immunocytochemistry and quantitative (real-time) RT-PCR.…”

“…The B27 supplement was frequently used for neural stem cell cultures and for neural differentiation of somatic stem cells (Engelhardt et al, 2004(Engelhardt et al, , 2005Miura et al, 2003;Wachs et al, 2003;Widera et al, 2007). NSCM II was a two-step neural differentiation protocol adapted from a successful protocol for neural differentiation of mesenchymal stem cells (Hermann et al, 2004(Hermann et al, , 2006.…”

“…Shi and coworkers isolated SHED (stem cells from human exfoliated deciduous teeth) that can differentiate into a variety of cell types, including neural cells (Miura et al, 2003). Recently, neural stem cells could be isolated from dental tissues, which could be propagated as neurospheres under serum-free medium conditions supplemented with growth factors (Widera et al, 2007). Today, the formation of neurospheres is the most common way of isolating and expanding neural stem cells in vitro .…”

“…Today, the formation of neurospheres is the most common way of isolating and expanding neural stem cells in vitro . Isolated periodontal stem cells were highly proliferative (Widera et al, 2007). Growth factor deprivation and retinoic acid treatment led to the acquisition of neural morphology and stable expression of markers of neural differentiation by more than 90% of the cells (Widera et al, 2007).…”

“…Isolated periodontal stem cells were highly proliferative (Widera et al, 2007). Growth factor deprivation and retinoic acid treatment led to the acquisition of neural morphology and stable expression of markers of neural differentiation by more than 90% of the cells (Widera et al, 2007). In contrast to these periodontalderived neural stem cells, dental pulp-derived stem cells or dental follicle cells (DFCs) are -like bone marrow-derived mesenchymal stem cells -plastic-adherent cells.…”