Vasculogenic Precedes Neurogenic Differentiation in Dental Pulp Stem Cells

Dental pulp stem cells (DPSCs) are neural crest-derived stem cells endowed with multipotency and self-renewal. While processes orchestrating DPSC differentiation have been studied extensively, mechanisms underpinning the differentiation of human DPSCs <i>in vivo</i> remain unclear. Here, we induced vasculogenic, odontoblastic, or neurogenic differentiation of human DPSCs for 7 d <i>in vitro</i> and performed single-cell RNA sequencing. Then, human DPSCs tagged with green fluorescent protein (DPSC-GFP) seeded in human tooth slice/scaffolds were transplanted into the subcutaneous space of immunodeficient mice. DPSC-GFP were sorted by flow cytometry 7 and 21 d after transplantation, and single-cell RNA sequencing was performed. In addition, a time course study was performed to investigate the sequence of differentiation events triggered upon transplantation of DPSC-GFP into mice. Here, we observed 8 distinct clusters of DPSCs at baseline, indicating a high level of cell heterogeneity. When DPSCs were induced to undergo vasculogenic, odontoblastic, or neurogenic differentiation <i>in vitro</i>, we observed distinct shifts in patterns of gene expression. Although some DPSCs retained mesenchymal stem cell markers likely due to asymmetric cell division and self-renewal, each differentiation protocol resulted in a unique gene expression signature. Stem cell markers that were highly expressed in DPSCs pretransplantation were progressively downregulated after 7 and 21 d in vivo. In contrast, endothelial cell markers presented high expression levels 7 d after transplantation, while neuronal markers showed upregulation 21 d after transplantation. Notably, while DPSC-derived functional blood vessels (i.e., blood-carrying vessels) can be clearly seen 2 wk after transplantation, well-defined DPSC-derived neural structures can be observed only after 5 wk. In conclusion, DPSCs are heterogeneous stem cells with distinct cell clusters, all of which contain progenitor cells with unique differentiation potential. Furthermore, this work demonstrated that microenvironment cues generated within human root canals are sufficient to induce vasculogenic differentiation, followed by neurogenic differentiation of DPSCs <i>in vivo</i>.