Time-course of endothelial differentiation by self-assembly of dental stem cells

Abstract

Objectives: The current study aimed to investigate the time-course effects on endothelial differentiation of dental stem cells, when they are allowed to self-assemble into microtissues. Methods: Micro-molds purchased from Microtissues, Inc. were used to fabricate agarose three-dimensional (3D) petri-dishes, which contain circular recesses. Dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) suspensions were poured into these petri-dishes and allowed to self-assemble into 3D microtissue spheroids over 24 hours. After 24 hours, medium was changed to fully supplemented endothelial growth medium -2 (Lonza Biologics Inc.) and subsequently changed in every three days. After 3-, 6- and 9- days, microtissue spheroids were transferred onto the two-dimensional culture dishes and allowed to attach and dissociate into single cells. At confluence, cells were trypsinized and examined for expression of endothelial markers – vWF, CD31, eNOS, VE-cadherin, VEGFR-1 and 2 via qPCR, western blotting and immunofluorescence. The capacity of these cells to form in-vitro capillary-like tubes on basement membrane matrix – Matrigel was assessed. Results: qPCR and immunofluorescence results showed a significantly higher levels of expression of endothelial markers in 3-days induced, microtissue-derived DPSCs and SHED compared to that of 6- and 9- days induced cells. Accordingly, 3-days induced, microtissue-derived SHED were able to form extensive capillary-like tubes on Matrigel, while cells derived from microtissues induced for 6- and 9- days failed to form an extensive tube-network. In contrast, microtissue-derived DPSCs failed to form capillary-like tubes regardless of the duration of induction. Both DPSCs and SHED, when cultured in traditional two-dimensional cultures did not show a significant increase in endothelial marker expression or capillary-like tube formation on Matrigel regardless of the duration of induction. Conclusions: SHED hold a higher potential for endothelial differentiation, when they were self-assembled into 3D spheroids and induced for 3 days compared to that of DPSCs.