Establishment and neural differentiation of neural crest‐derived stem cells from human dental pulp in serum‐free conditions

The potential of obtaining cell cultures with neural crest resemblance (neural crest‐derived stem cells [NCSCs]) from dental‐related tissues, including human dental pulp cells (hDPCs), has been discussed in the literature. However, most reports include the use of serum‐rich conditions and do not describe the potential for neural differentiation, slowing translation to the clinic. Therefore, we aimed to culture and characterize NCSCs from the human dental pulp in vitro and evaluate their ability to differentiate into neurons; we also investigated the effectiveness of the addition of BMP4 to enhance this potential. Cultures were established from a varied cohort of patient samples and grown, as monolayers, in serum, serum‐free, and also under sphere‐aggregation conditions to induce and identify a NCSC phenotype. hDPC cultures were characterized by immunocytochemistry and reverse transcription quantitative polymerase chain reaction. Monolayer cultures expressed stem cell, neural progenitor and neural crest‐related markers. Culturing hDPCs as neurospheres (hDPC‐NCSCs) resulted in an increased expression of neural crest‐related genes, while the addition of BMP4 appeared to produce better NCSC characteristics and neural differentiation. The neural‐like phenotype was evidenced by the expression of TUJ1, peripherin, NFH, TAU, SYN1, and GAP43. Our results describe the establishment of hDPC cultures from a large variety of patients in serum‐free medium, as NCSC that differentiate into neural‐like cells, as well as an important effect of BMP4 in enhancing the neural crest phenotype and differentiation of hDPCs. Human dental pulp tissue collected from a varied cohort of samples was used to directly establish monolayer cultures in defined serum‐free conditions. In monolayer, the cultures maintained an mesenchymal stem cell (MSC) phenotype regardless of the initial serum‐free conditions. A Neural crest‐derived stem cell phenotype was favored by adding BMP4 before or after transferring the cells to a 3D culture.