Single-cell RNA sequencing reveals vascularization-associated cell subpopulations in dental pulp: PDGFRβ plus DPSCs with activated PI3K/AKT pathway

Background This study aims to address challenges in dental pulp regeneration therapy. The heterogeneity of DPSCs poses challenges, especially in stem cell transplantation for clinical use, particularly when sourced from donors of different ages and conditions.Methods Pseudotime analysis was employed to analyze single-cell sequencing data, and immunohistochemical studies were conducted to investigate the expression of fibronectin 1 (FN1). We performed in vitro sorting of PDGFR beta+ DPSCs using flow cytometry. A series of functional assays, including cell proliferation, scratch, and tube formation assays, were performed to experimentally validate the vasculogenic capabilities of the identified PDGFR beta+ DPSC subset. Furthermore, gene-edited mouse models were utilized to demonstrate the importance of PDGFR beta+ DPSCs. Transcriptomic sequencing was conducted to compare the differences between PDGFR beta+ DPSCs and P1-DPSCs.Results Single-cell sequencing analysis unveiled a distinct subset, PDGFR beta+ DPSCs, characterized by significantly elevated FN1 expression during dental pulp development. Subsequent cell experiments demonstrated that this subset possesses remarkable abilities to promote HUVEC proliferation, migration, and tube formation. Gene-edited mouse models confirmed the vital role of PDGFR beta+ DPSCs in dental pulp development. Transcriptomic sequencing and in vitro experiments demonstrated that the PDGFR/PI3K/AKT signaling pathway is a crucial factor mediating the proliferation rate and pro-angiogenic properties of PDGFR beta+ DPSCs.Conclusion We defined a new subset, PDGFR beta+ DPSCs, characterized by strong proliferative activity and pro-angiogenic capabilities, demonstrating significant clinical translational potential. Graphical Abstract