VEGF quantification and HUVECs tube formation assay

The culture supernatant of control and treatment groups was collected from the in vitro coculture system after 14 days and assayed to measure the level of VEGF. A human VEGF ELISA kit was used to quantify VEGF in medium from HASCs and HASCs/HAMSCs groups, according to the manufacturer’s instructions. The measured values were expressed as fold changes over that of the control: HASCs treated without HAMSCs.

HUVECs were subjected to the culture supernatant from control and treatment groups to assay the formation of tube-like structures. Six-well culture plates were coated with Matrigel according to the manufacturer’s instructions. After HUVECs were incubated in EBM containing 1% FBS and EGM-2 BulletKit for 6 h and plated onto the layer of Matrigel at a density of 5×10⁴ cells/well, medium was replaced by the culture supernatant from HASCs and HASCs/HAMSCs groups. Matrigel cultures were incubated at 37 C for 24 h. Following rinsing with PBS, tube formation was visualized using an inverted microscope (Carl Zeiss AG, Oberkochen, Germany) and representative network of formed tube structures was randomly photographed five shots per each group.