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 postimplantation 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 short hairpin RNA. HIF-1α–stabilized SHED were encapsulated in PuraMatrix hydrogel, injected into root canals of human tooth fragments, and implanted in the subcutaneous space of immunodeficient mice. After 28 d, 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 odontogenic/osteogenic differentiation both in vivo and in vitro were observed. At 7 d postimplantation, 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 and 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, which contributes to the maintenance of reactive oxygen species homeostasis and metabolic adaptation in hypoxia. In addition, we identified Smad7 as 1 of the top 3 upregulated genes through RNA sequencing 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, as well as odontogenic tissue formation during dental pulp regeneration, which could benefit stem cell–based therapy in general.

基于干细胞的疗法是牙髓再生的一种有前途的策略。然而，由于缺血性微环境，体内移植后的低细胞活力仍然是未来临床应用的关键挑战。为了提高植入后细胞的存活率和牙髓组织再生，来自人类脱落乳牙 (SHED) 的干细胞通过含脯氨酰羟化酶结构域的蛋白质的敲低，通过缺氧诱导因子 1α (HIF-1α) 稳定化预处理至缺氧状态2 (PHD2) 使用慢病毒短发夹 RNA。HIF-1α 稳定的 SHED 被封装在 PuraMatrix 水凝胶中，注射到人类牙齿碎片的根管中，并植入免疫缺陷小鼠的皮下空间。28 d 后，观察到牙髓样组织形成增强，血管化水平显着提高，这可能归因于 SHED 的内皮分化和宿主血管的募集。此外，观察到体内和体外的牙本质样组织形成和加速的成牙/成骨分化。在植入后 7 天，与对照 SHED 相比，在 HIF-1α 稳定的 SHED 组中检测到显着更少的 DNA 损伤和更高的 Ki67 表达。因此，体外Ki67和凋亡细胞的细胞活力测定和染色表明，HIF-1α稳定可以显着减少细胞凋亡并提高细胞存活率。我们证明了 PI3K/AKT 通路激活导致缺氧条件下 HIF-1α 稳定的 SHED 中 caspase 3 表达低。此外，我们发现 HIF-1α 诱导的细胞存活也可归因于 PDK1、HK2 和 Glut1 的上调表达，这有助于维持活性氧稳态和缺氧时的代谢适应。此外，我们确定了通过在 HIF-1α 稳定的 SHED 中通过 RNA 测序，Smad7作为前 3 个上调基因之一，并证明了其在 HK2 和 Glut1 上调中的重要作用。总之，HIF-1α 稳定性通过调节各种靶基因和潜在信号通路以及牙髓再生过程中牙源性组织的形成来提高 SHED 的细胞存活率，这通常有利于基于干细胞的治疗。