Supporting data for "HIF-1α Stabilization Promotes Pulp Regeneration by Enhancing Angio-/Vasculogenesis and Survival of Dental Stem Cells"

Stem cell-based therapeutics is a promising strategy in dental pulp regeneration. However, low cell viability after transplantation in-vivo due to the ischemic microenvironment is still a critical challenge for future clinical application. With the aim of improving post-implantation cell survival and pulp tissue regeneration, stem cells from human exfoliated deciduous teeth (SHED) were preconditioned to a hypoxic condition by hypoxia-inducible factor-1α (HIF-1α) stabilization via knockdown of prolyl hydroxylase domain-containing protein 2 (PHD2) using lentiviral-shRNA. HIF-1α-stabilized-SHED were encapsulated in PuraMatrixTM hydrogel, injected into root canals of human tooth fragments, and implanted in the subcutaneous space of immunodeficient mice. After 28-days, enhanced dental pulp-like tissue formation was observed with a significantly higher level of vascularization, which could be attributed to both endothelial differentiation of SHED and recruitment of host blood vessels. Furthermore, dentin-like tissue formation in-vivo and accelerated odonto-/osteogenic differentiation both in-vivo and in-vitro were observed. At 7-days post-implantation, significantly less DNA damage and higher ki67 expression were detected in the HIF-1α-stabilized-SHED group compared with the Control-SHED. Accordingly, cell-viability assay and staining for ki67 apoptotic cells in-vitro showed that HIF-1α stabilization could decrease cell apoptosis and enhance cell survival significantly. We demonstrated that PI3K/AKT pathway activation had resulted in low caspase-3 expression in HIF-1α-stabilized-SHED in hypoxic conditions. Furthermore, we found that HIF-1α induced cell survival could also be attributed to the upregulated expression of  PDK1, HK2, and Glut1 genes, which contributes to the maintenance of ROS homeostasis and metabolic adaptation in hypoxia. Additionally, we identified Smad7 as one of the top 3 upregulated genes through RNA-seq in HIF-1α-stabilized-SHED  and demonstrated its essential role in HK2 and Glut1 upregulation. Taken together, HIF-1α stabilization enhances cell survival of SHED through modulating various target genes and potential signaling pathways, and odontogenic tissue formation during dental pulp regeneration, which could benefit stem cell-based therapy in general.

基于干细胞的再生治疗策略在牙髓再生领域具有广阔的应用前景。然而，由于缺血性微环境导致的移植后细胞存活率低，这仍然是未来临床应用中亟待克服的关键挑战。为了提高植入后细胞的存活率和牙髓组织的再生，通过对脯氨酰羟化酶结构域蛋白2（PHD2）的敲低，利用缺氧诱导因子-1α（HIF-1α）的稳定化处理，将人脱落的乳牙干细胞（SHED）预先置于缺氧状态。经过HIF-1α稳定的SHED被封装于PuraMatrix™水凝胶中，注入到人牙碎片根管内，并在免疫缺陷小鼠的皮下空间进行植入。经过28天的观察，发现牙髓样组织形成得到增强，血管化水平显著提高，这可能是由于SHED的血管内皮细胞分化和宿主血管的募集共同作用。此外，还观察到在体和离体条件下牙本质样组织的形成以及牙源性和成骨性分化的加速。在植入后第7天，与对照SHED组相比，HIF-1α稳定的SHED组检测到显著较低的DNA损伤和较高的Ki67表达。相应地，体外细胞活力检测和Ki67凋亡细胞染色显示，HIF-1α的稳定化处理可以显著降低细胞凋亡并增强细胞存活。本研究证明，PI3K/AKT途径的激活导致了HIF-1α稳定的SHED在缺氧条件下的caspase-3表达降低。此外，我们还发现HIF-1α诱导的细胞存活也可以归因于PDK1、HK2和Glut1基因的上调表达，这些基因有助于维持缺氧条件下的ROS稳态和代谢适应。此外，我们通过RNA测序发现Smad7是HIF-1α稳定的SHED中上调表达的前三位基因之一，并证明了其在HK2和Glut1上调中的关键作用。综上所述，HIF-1α的稳定化通过调节多种靶基因和潜在信号通路，增强了SHED的细胞存活能力，并在牙髓再生过程中促进了牙源组织的形成，这有望为基于干细胞的疗法带来普遍的益处。