Mechanisms of dental pulp stem cell differentiation into functional endothelium

项目摘要/摘要 问题：人类牙髓含有间充质干细胞，即牙髓干细胞 协调牙齿发育和组织再生。在第一个资金周期中，我们展示了DPSC是 能够分化成内皮细胞，形成功能性血管。这些发现表明， 多能DPSC可以分化为组织形成细胞(如成牙本质细胞)，同时显示出 生成血管以支持组织再生的代谢需求。然而，这些机制 DPSC用来生成血管的机制尚不清楚，因此我们利用这种分化的能力也不清楚 这些细胞在治疗组织再生方面的潜力是有限的。假设：我们已经证明了信号 由血管内皮细胞生长因子(VEGF)通过其受体VEGFR1启动，并由 WNT/B-catenin通路，调节DPSC的内皮分化。在初步研究中，我们做出了 令人兴奋的观察到DPSC来源的血管与宿主血管吻合并形成 由平滑肌细胞/周细胞投资。然而，监管这些事件的机制尚不清楚。 血管内皮细胞黏附蛋白在血管内皮细胞定位、血管组织和吻合中起关键作用 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过程中发挥作用。 以牙髓干细胞作为MSC的实验模型。 此外，人们还推测，通过MSC再生的组织的长期存活率和功能 取决于这些细胞重建和维持干细胞生态位的能力。初步数据显示 内皮细胞衍生因子诱导DPSC自我更新，如Bmi-1上调和 二次球体编队。在这里，我们将探索干细胞因子(SCF)/c-Kit轴在 干细胞在DPSC再生组织中的维持。我们的工作假说是：“血管生成 牙髓干细胞通过内皮细胞分化与宿主血管吻合，在壁上成熟 细胞投资，并维持干细胞池“。为了验证这一假设，我们提出了以下具体建议 目的：S.A.#1：确定VE-钙粘附素在DPSC来源的血管吻合中的作用；S.A.#2： 确定血管生成素信号在DPSC来源的血管成熟中的作用；S.A.#3： 明确SCF/c-Kit通路在DPSC产生的组织中维持干细胞的作用。 意义：干细胞疗法的临床翻译需要对机制的理解 它们控制着这些细胞的分化命运。这一项目的成功完成将导致机制-- 开发间充质干细胞在组织再生中的血管生成潜力的基础疗法。这个 开发安全的策略，使移植后产生的组织能够有效地血管化 干细胞移植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.