DOI: 10.25442/hku.20726935.v1
title: Supporting dataset for "SEMA4D-PLEXIN-B1 SIGNALING IN VASCULAR STABILIZATION BY RECRUITMENT OF DENTAL STEM CELLS"

description: Vascularization is one of the key challenges in tissue regeneration. Nascent vessels formed by endothelial cells in initiation stage are immature and unstable. The recruitment of mural cells, such as pericytes or vascular smooth muscle cells, is critical for stabilization of nascent vessels. Stem cells from human exfoliated deciduous teeth (SHED) are considered to have mural cell-like properties. However, the signaling mechanisms that regulate the cross-talk between endothelial cells and SHED in recruiting them as mural cells is much less well understood. This study aimed to examine the role of Semaphorin 4D (Sema4D)-Plexin-B1 signaling in the recruitment of SHED as mural cells to stabilize the EC formed vascular tubes <em>in vitro </em>and <em>in vivo</em>. Herein, using a 3D biomimetic microfluidic device, for the first time, we unraveled the role of Sema4D-Plexin-B1 signaling in the recruitment of SHED as mural cells during angiogenic sprouting and vasculature formation by endothelial cells in a 3D fibrin matrix. The specific compartmentalized design of the microfluidic chip facilitated to recreate the multi-step dynamic process of angiogenesis in a time and space dependent manner. The results showed that Sema4D significantly enhanced the vessel network formation and the percentage of SHED-covered vascular structures. Trans-well assay showed that Sema4D increased the migration of SHED through inducing the secretion of endothelial-derived factors. Furthermore, Sema4D-induced paracrine signaling transformed SHED into a more mature mural phenotype by increasing the expression of NG2, α-SMA, and SM22α. Our results further demonstrated that Sema4D exerts these effects by acting on endothelial- Plexin-B1 by inducing expression of platelet derived growth factor (PDGF)-BB, which is a major mural cell recruitment factor. The <em>in vivo </em>Matrigel plug assay indicated that Sema4D added group showed the highest levels of vascularization at 7 and 14 days of implantation. Besides, Sema4D significantly increased the number of stabilized vessels lined by SM22α positive SHED (SM22α+SHED). In addition to SM22α, IF staining for PDGFR-b and NG2 was performed to demonstrate the mural cell-like activity of SHED. In accordance with the <em>in vitro </em>results, we demonstrated that Sema4D exerts its effects on human umbilical vein endothelial cells (HUVECs) through its high affinity receptor Plexin-B1 as shown by the lack of mural cell recruitment in the presence of Sema4D with Plexin-B1 knocked down endothelial cells. Accordingly, when PDGFR-β was blocked, the effects of Sema4D on the recruitment of SHED as mural cells was significantly reduced. In the current study, we demonstrated that Sema4D facilitates the interaction between endothelial cells and SHED and promotes the recruitment of SHED as mural cells in vascular stabilization. The results of this study further highlight the potential role of Sema4D as a target growth factor in fabrication of engineered tissue constructs with mature vessels.