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

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posted on 2023-04-26, 01:25 authored by Yuanyuan HanYuanyuan Han, Waruna Lakmal DissanayakaWaruna Lakmal Dissanayaka

  

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.

Funding

Enhancing post-implantation dental stem cell survival and angiogenesis via in vitro stabilization of HIF-1α signaling

University Grants Committee

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The Hif-1α-SEMA4D-Plexin-B1 signaling axis regulates dental stem cells in the stabilization of vascular structures

University Grants Committee

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