Vasculogenic Precedes Neurogenic Differentiation in DPSC Transplanted into Root Canals

Dental pulp stem cells (DPSC) constitute a neural crest-derived stem cell population endowed with multipotency and self-renewal. While the process of DPSC differentiation has been studied extensively in vitro, very little is known about mechanisms underpinning the differentiation of human DPSC in vivo. Here, we induced vasculogenic, odontoblastic, or neurogenic differentiation of human DPSC for 7 days in vitro and performed single cell sequencing. Then, human DPSC tagged with GFP (DPSC-GFP) seeded in human tooth slice/scaffolds were transplanted into immunodeficient mice. Single cell sequencing of DPSC-GFP sorted by flow cytometry was performed 7 and 21 days after transplantation. We observed major shifts in patterns of gene expression when DPSC were induced to undergo vasculogenic, odontoblastic, or neurogenic differentiation in vitro. Although some DPSC retained mesenchymal stem cell (MSC) markers (indicating asymmetric cell division and self-renewal), each differentiation protocol resulted in a unique gene expression signature in vitro. In vivo, stem cell markers that were highly expressed in DPSC pre-transplantation gradually decreased in expression after 7 and 21 days. In contrast, vascular endothelial cell markers were highly expressed 7 days after transplantation, while neuronal markers were highly expressed 21 days after transplantation. In conclusion, DPSC cells are heterogeneous with clearly distinct cell clusters, all of which contain cells with unique differentiation potential. Notably, the microenvironment created when human DPSC are transplanted inside a human root canal in vivo induces vasculogenic differentiation first, which is then followed by neurogenic differentiation. Overall design: RNS-seq profiles of dental pulp stem cells before and after differentiation into three different cell types (angiogenic, neurogenic, and odontogenic), and RNA-seq profiles of DPSCs extracted 7 and 21 days after subcutaneous transplantation into mice.