Mechanisms of dental pulp stem cell differentiation into functional endothelium

项目概要/摘要 问题：人类牙髓含有间充质干细胞（MSC），即牙髓干细胞（DPSC） 协调牙齿发育和组织再生。在第一个融资周期中，我们表明 DPSC 能够分化成形成功能性血管的内皮细胞。这些发现表明 多能 DPSC 可以分化为组织形成细胞（例如成牙本质细胞），同时表现出 产生血管来支持组织再生的代谢需求。然而，这些机制 DPSC 用于生成血管的方式尚不清楚，因此我们利用分化的能力 这些细胞在治疗性组织再生中的潜力是有限的。假设：我们已经证明信号传导 由血管内皮生长因子 (VEGF) 通过其受体 VEGFR1 启动，并由经典 Wnt/B-catenin通路，调节DPSC的内皮分化。在初步研究中，我们做了 令人兴奋的观察结果是，DPSC 衍生的血管与宿主脉管系统吻合并成为 由平滑肌细胞/周细胞投资。然而，调节这些事件的机制尚不清楚。 VE-钙粘蛋白在血管内皮细胞定向、血管组织和吻合过程中发挥关键作用 胚胎发育。一旦血管形成，血管生成素/Tie2通路就会调节血管 成熟。在这里，我们将确定这些信号通路是否在吻合和成熟中发挥作用 出生后干细胞来源的血管，使用牙髓干细胞作为 MSC 的实验模型。 此外，据推测，用 MSC 再生的组织的长期活力和功能 取决于这些细胞重建和维持干细胞生态位的能力。初步数据表明 内皮细胞衍生因子诱导 DPSC 自我更新，如 Bmi-1 上调和 二次球体的形成。在这里，我们将探讨干细胞因子 (SCF)/c-Kit 轴在 维持 DPSC 再生组织中的干细胞。我们的工作假设是：“血管产生 牙髓干细胞通过内皮分化与宿主脉管系统吻合，在壁上成熟 细胞投资，并维持干细胞库”。为了验证这一假设，我们提出以下具体方案 目标：S.A.#1：定义 VE-钙粘蛋白在 DPSC 来源血管吻合中的作用； SA#2： 明确血管生成素信号传导对 DPSC 来源血管成熟的作用； S.A.#3：至 定义了 SCF/c-Kit 通路在 DPSC 生成组织中干细胞维持中的作用。 意义：干细胞疗法的临床转化需要了解机制 控制这些细胞的分化命运。该项目的成功完成将导致机制—— 基于间充质干细胞在组织再生中的血管生成潜力的疗法。的 开发安全策略，使移植后产生的组织能够有效地血管化 干细胞将使大量需要治疗性组织再生的患者受益。

PROJECT SUMMARY/ABSTRACT The Problem: Human dental pulps contain mesenchymal stem cells (MSC), i.e. dental pulp stem cells (DPSC) that orchestrate tooth development and tissue regeneration. In the 1st funding cycle, we showed that DPSC are capable of differentiating into endothelial cells that form functional blood vessels. These findings suggest that pluripotent DPSC can differentiate into tissue-forming cells (e.g. odontoblasts) while exhibiting the capacity to generate blood vessels to support the metabolic demands of tissue regeneration. However, the mechanisms employed by DPSC to generate blood vessels are unclear, and therefore our ability to exploit the differentiation potential of these cells in therapeutic tissue regeneration is limited. Hypothesis: We have shown that signaling initiated by vascular endothelial growth factor (VEGF) through its receptor VEGFR1, and by the canonical Wnt/B-catenin pathway, regulate the endothelial differentiation of DPSC. In preliminary studies, we made the exciting observation that DPSC-derived blood vessels anastomize with the host vasculature and become invested by smooth muscle cells/pericytes. However, the mechanisms regulating these events are not known. VE-Cadherin plays a key role in endothelial cell orientation, blood vessel organization and anastomosis during embryonic development. Once blood vessels are formed, the Angiopoietin/Tie2 pathway regulates vessel maturation. Here, we will determine if these signaling pathways play a role in the anastomosis and maturation of post-natal stem cell-derived blood vessels, using dental pulp stem cells as experimental models of MSC. Furthermore, it has been postulated that the long-term viability and function of tissues regenerated with MSC depends on the ability of these cells to reconstitute and maintain stem cell niches. Preliminary data suggest that endothelial cell-derived factors induce self-renewal of DPSC, as demonstrated by Bmi-1 upregulation and secondary sphere formation. Here, we will explore the role of the Stem Cell Factor (SCF)/c-Kit axis on the maintenance of stem cells in DPSC-regenerated tissues. Our working hypothesis is: “Blood vessels generated by endothelial differentiation of dental pulp stem cells anastomize with the host vasculature, mature upon mural cell investment, and maintain a pool of stem cells”. To test this hypothesis, we propose the following specific aims: S.A.#1: To define the role of VE-Cadherin on the anastomosis of DPSC-derived blood vessels; S.A.#2: To define the function of Angiopoietin signaling on the maturation of DPSC-derived blood vessels; S.A.#3: To define the role of the SCF/c-Kit pathway in the maintenance of stem cells in DPSC-generated tissues. Significance: The clinical translation of stem cell-based therapies requires the understanding of mechanisms that control the differentiation fate of these cells. Successful completion of this project will lead to mechanism- based therapies that exploit the vasculogenic potential of mesenchymal stem cells in tissue regeneration. The development of safe strategies that enable efficient vascularization of tissues generated upon transplantation of stem cells will benefit a large number of patients that require therapeutic tissue regeneration.