Differentiation of dental pulp stem cells into neuron-like cells in serum-free medium

Abstract

Dental pulp tissue contains dental pulp stem cells (DPSCs). Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells) are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146(+) , Cd166(+) , and Cd31(-) in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

Figure 1

Characteristics of isolated and in vitro mouse dental pulp stem cells. Colonies derived from dental pulp at the first passage (a) and after 24 hours of culture (b). Colonies began to show changes in shape after the second passage (c), a fibroblastic cell shape after the fourth passage (d), and confluence after 2-3 days of culture in complete medium (e).

Figure 2

Activation of mesenchymal stem cell markers. The activation of Cd146 (~479 bp) (a) and Cd166 (~630 bp) (b) was observed only in cells before differentiation, indicating that the cells were mesenchymal stem cells. Cd31 was inactivated before and after neuronal differentiation (c). Gapdh (~717 bp), a housekeeping gene, was expressed before and after differentiation (d). Panel (i) is representing undifferentiated cells, that is, cells before neuronal differentiation, while panel (ii) is representing differentiated cells, that is, cells after neuronal differentiation.

Figure 3

Characteristics of differentiated cells. Neuron-like cells appeared among the dental pulp stem cells ((a), (b)) 5 days after neuronal induction (c). The perikaryon, dendrites, and axons of neurons are indicated, respectively, by white arrows, open arrowheads, and black arrows (d). Immunofluorescence staining for neuron markers MAP2 was performed after 5 days of neuronal induction ((e), (f)). MAP2 marker was not detected in DPSCs without neuronal induction, that is, negative control (g).

Figure 4

Activation of specific neuronal markers. The activation of Nestin (~215 bp) (a) and Tub3 (~125 bp) (b) indicated that the cells had differentiated into neurons. Gapdh (~717 bp), a housekeeping gene, was activated before and after differentiation (c). Panel (i) is representing undifferentiated cells, that is, cells before neuronal differentiation, while panel (ii) is representing differentiated cells, that is, cells after neuronal differentiation.

Figure 5

Viability of cells during directed differentiation. The viability of control (undifferentiated cells cultured in complete medium) versus differentiated cells cultured in serum- and growth factor-free medium. The numbers of both control and differentiated cells were significantly increased upon differentiation compared with day 0 of culture. The results are summarized as the mean ± SD. Statistical significance was determined using SPSS program version 16.0.2. *Statistical analysis showed significant differences (P < 0.05) of viable cells at days 3 and 5 of culture as compared to control.