HIF-1α stabilized SHED in 3D spheroids promote endothelial differentiation and improve hindlimb ischemia

Abstract

Peripheral artery disease (PAD) is a type of vascular disease (VD) that can cause severe limb ischemia, with the most common type being lower limb ischemia. Critical lower limb (CLI) ischemia represents the end stage of the condition. Despite attempts to use various types of stem cells to treat CLI, their effectiveness remains limited. There is still a need to study how to effectively differentiate mesenchymal stem cells into endothelial cells (ECs) and utilize them to promote vasculature regeneration. This study aims to promote the differentiation of stem cells from human deciduous teeth (SHED) into ECs through hypoxia-inducible factor 1α (HIF-1α) stabilization and 3D spheroid culture and to use these induced cells to improve CLI. HIF-1α stabilization in SHED was achieved by silencing prolyl hydroxylase domain-containing protein 2 (PHD2) via target small hairpin RNA (shRNA). HIF- 1α-stabilized-SHED were allowed to self-assemble into spheroids on microwells of agar Petri dishes, cultured for 7 days, and were transferred onto two-dimensional culture dishes and cultured for 7 days to obtain induced-SHED. The proliferation of transfected cells was assessed by CCK-8 assay. Western blotting for PHD2, HIF- 1α, and VEGFR2, CD31 was performed to evaluate transfection efficiency and determine the optimal spheroid diameter, respectively. Endothelial differentiation was assessed by the expression of EC-specific markers as detected via RT-PCR, western blotting, and immunofluorescence. Spheroidal sprouting assay and Matrigel assay were performed to assess the functionality of induced SHED. RNA- sequencing was carried out to analyze the transcriptional pathways activated in induced SHED. Trans-well assay and immunofluorescence for endothelial matrix markers laminin and vimentin were performed to characterize the induced SHED further. Induced-SHED were encapsulated in fibrin hydrogel and injected into CLI mice. Ischemia scores and blood perfusion records were used to measure the extent of functional recovery in CLI mice. Immunohistochemistry and Masson’s trichrome stain evaluated the effects of cell therapy on tissue regeneration. After PHD2 knockdown, HIF-1α protein expression was significantly increased without affecting cell proliferation. Spheroids of HIF-1α-stabilized-SHED with a diameter of 400 μm were identified as optimal to promote endothelial

differentiation. The expression of EC-markers VEGFR2 and CD31 in HIF-1α- stabilized-SHED spheroids was significantly increased in both mRNA and protein levels, and the sprouting length was significantly increased in fibrin gel assay. HIF- 1α-stabilized spheroid-derived SHED (Induced-SHED) not only maintained a high level of EC-marker expression but also formed vascular tubes that are similar to that of HUVECs on Matrigel. RNA-sequencing results demonstrated significant up- regulation of genes and pathways associated with ECs and angiogenesis. Furthermore, the migration ability and the expression of ECM-specific markers of Induced-SHED were also significantly improved. In an in vivo experiment, Induced SHED not only significantly enhanced blood perfusion, angiogenesis, and neovascularization but also alleviated tissue fibrosis in CLI mice. In conclusion, HIF-1α stabilization and 3D spheroid culture could effectively promote the endothelial differentiation and angiogenic properties of SHED and ultimately improve the hindlimb ischemia of mice, which provides a promising approach for the utilization of SHED in the treatment of VD.