Endothelial differentiation of dental stem cells in a three-dimensional microtissue culture system

Abstract

Dental stem cells have been demonstrated to have potential to differentiate into several lineages, including endothelial cells. However, the efficiency of in-vitro endothelial differentiation of dental pulp stem cells (DPSCs) and stem cells from exfoliated human deciduous teeth (SHED) is questionable. The present study aimed to investigate the endothelial differentiation potential of DPSCs and SHED in a three-dimensional microtissue spheroid system. Threedimensional (3D) microtissue-spheroids of DPSCs and SHED were fabricated using 12-series micro-molds (MicroTissues Inc.). DPSCs and SHED cultured on two-dimensional (2D) sixwell culture plates were used as the control groups. Twentyfour hours after DPSCs and SHED were seeded in plates (2D) or micro-molds (3D), the culture medium was changed to fully supplemented endothelial growth medium -2 (Lonza Biologics Inc.) After 3-, 6-, and 9- days of induction, 3D microtissues were transferred onto the adherent culture surfaces and allowed to dissociate into cells, while 2D cultures were trypsinized and sub-cultured. Then, the cells were analyzed for expression of endothelial markers – eNOS, vWF, CD31, VE-cadherin, VEGFR-1 and 2 via immunofluorescence and qPCR. Matrigel assay was performed to assess the cells’ ability to form capillary-like tube structures in-vitro. Immunofluorescence and qPCR results indicated an enhanced endothelial differentiation of 3D microtissue derived cells compared with 2D induced cells in relation to both DPSCs and SHED. SHED dissociated from induced microtissues were positive for endothelial markers and were able to form endothelial-like tube structures on matrigel. In contrast, 2D induced SHED failed to form such structures on Matrigel. However, DPSCs induced in either 3D or 2D could not form endothelial-like tube structures on matrigel. Threedimensional microtissue is a promising culture system for promoting endothelial differentiation efficiency of dental stem cells in-vitro. SHED hold a higher potential for endothelial differentiation compared to that of DPSCs. Funding Source Small Project Funding, HKU. (Project code: 201409176177