Enhanced regenerative potential of human dental pulp stem cells for the pulp-dentin complex through coculture with iPSC-derived endothelial cells: An in vitro study

Abstract

Objectives: Although cell-based therapies using human dental pulp stem cells (hDPSCs) with other cell lineages and growth factors show promise in regenerative endodontics, combining hDPSCs with induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) is unexplored. Moreover, iPSC-ECs overcome ethical and practical challenges related to primary endothelial cells. This study explored the odontogenic and angiogenic potential of hDPSCs and iPSC-ECs in direct coculture.

Design: hDPSCs were isolated from extracted human teeth, and iPSC‑ECs were generated via episomal reprogramming of hDPSCs followed by endothelial differentiation. Four groups were established for differentiation assays: hDPSCs in basal medium, osteogenic medium, modified osteogenic medium (D‑MOD), and coculture with iPSC‑ECs (1:5) in D‑MOD. Mineralization was assessed by alkaline phosphatase and alizarin red S staining; gene expression of odontogenic (DSPP, IBSP, ALPL) and angiogenic (PECAM1, MCAM, KDR) markers was measured by RT‑qPCR; protein levels were evaluated by Western blot and nestin immunofluorescence; and angiogenic capacity in the D‑MOD and coculture groups was quantified via Matrigel tube‑formation assay.

Results: The coculture group showed enhanced mineralization and significantly increased expression of DSPP, IBSP, and PECAM1. Protein analysis confirmed elevated DSPP and nestin levels. Tube formation assays revealed significantly more junctions, segments, and meshes in the coculture group.

Conclusions: This study demonstrated in vitro that coculturing hDPSCs with iPSC-ECs enhances both odontogenic and angiogenic differentiation compared to hDPSCs cultured alone. These findings highlight the potential of iPSC technology in regenerative endodontics and indicate a promising cell-based approach for future therapeutic applications.