SUMMARY/ABSTRACT The Problem: The long-term outcome of tissues regenerated with stem cell-based therapies requires functional vascularization and enervation and a microenvironment that is supportive of the maintenance of undifferentiated stem cells. In the 1st funding cycle of this project, we demonstrated that neural crest-derived dental pulp stem cells (DPSC) can differentiate into vascular endothelial cells. In the 2nd cycle, we showed that DPSC-derived vessels mature upon investment by mural cells and anastomize with host vasculature to become fully functional blood vessels. During these studies, we observed that a sub-population of DPSCs functions as neural progenitor cells and differentiate into neural cells while other DPSCs remain undifferentiated via stem cell self-renewal. Collectively, these tightly modulated events enable the generation of neurovascular stem cell niches in tissues regenerated with DPSC. However, mechanisms underpinning the generation and maintenance of these niches are unclear. Therefore, our ability to engage the full potential of DPSCs in tissue regeneration is limited. Premise: It is known that Brain-Derived Neurotrophic Factor (BDNF) is critical for neurogenic differentiation of progenitor cells in the central nervous system (CNS) during embryonic development. In contrast, N-Cadherin was shown to maintain neural progenitor cells in an undifferentiated state in the CNS. Here, we will determine what is the role of these two signaling pathways in the balance between self-renewal (i.e. maintenance of stemness) and neurogenic differentiation of neural progenitor cells in the peripheral nervous system using DPSC as a prototypic mesenchymal stem cell (MSC) population. Further, using single cell RNA sequencing we found that untreated DPSCs are heterogeneous exhibiting 8 unique cell clusters. Here, we propose studies to understand how DPSC heterogeneity and plasticity impacts the generation and maintenance of neurovascular stem cell niches in the pulp. Our overall hypothesis is: “a balance between BDNF-induced neurogenic differentiation and N-Cadherin- induced self-renewal of neural progenitor cells enables the generation and maintenance of DPSC-derived neurovascular stem cell niches”. To test this hypothesis, we propose 3 specific aims: S.A.#1: To define the role of autocrine BDNF on the neurogenic differentiation of DPSC; S.A.#2: To define the role of N-Cadherin signaling on the maintenance of neural progenitor cells in DPSC-derived neurovascular stem cell niches; S.A.#3: To define the function of DPSC heterogeneity and plasticity on the generation and maintenance of neurovascular stem cell niches. Significance: Successful completion of this work will unveil mechanisms that orchestrate the generation of neurovascular stem cell niches. These niches create a microenvironment that supports the maintenance of undifferentiated stem cells that can be activated for replacement of dead terminally differentiated cells (e.g. odont...