Regulation of neural crest derived progenitor cell niche by epithelial cell derived extracellular vesicles

总体计划目标 该研究计划的总体目标是揭示调节口腔/牙周神经沟来源的基质干细胞(NCSSCs)的生态位和功能的机制。NCSSCs保留在少数特定的位置，如骨髓、毛囊和口腔粘膜中的成人组织，其数量通常随着年龄的增长而减少。利用WNT1-CRE/R26RYFP报告小鼠，我们通过示踪实验表明，牙龈NCSSCs在牙周结缔组织中丰富。有趣的是，NCSSCs集中在结缔组织乳头，更具体地说，是在靠近牙龈口腔上皮细胞(OECs)的地方。众所周知，结缔组织和上皮细胞参与了分子串扰，可以调节这两种细胞的功能。因此，OECs和NCSSCs之间的串扰可能参与了NCSSC生态位和功能的调节。文献中没有关于OEC如何调控NCSSC利基或命运的信息。在本研究中，我们提出了一种新的范式，即嗅鞘细胞释放参与调控NCSSC命运的胞外小泡。 一般假设 一般的假设是，来自邻近嗅鞘细胞的信号调节成年牙龈组织中NCSSCs的命运和生态位。因为信号需要是特异的，并且不受靶外效应的影响，我们进一步假设OECNCSSC的串扰是由细胞外小泡(EVS)调节的。包括外切体(EXO)和微囊泡(MVS)在内的EVS为这种调节提供了安全性和受体介导的特异性。 目标和方法 该提案的目的是了解嗅鞘细胞来源的EXOS和MVS如何调节NCSSCs中分化相关基因和蛋白的功能和表达。此外，我们将研究这些小泡如何调节特定的分化途径，包括成骨、成脂、成软骨和神经样谱系。我们将以我们从成人牙龈分离的NCSSC细胞株(命名为Ginestin，90%以上为巢蛋白阳性细胞)作为我们的靶细胞，将其暴露于OEC囊泡中，并用RT-qPCR、Western blotting、蛋白质组学、免疫染色等显微技术测量其产量。 预期结果和意义 这项研究计划有望显著增加我们对神经脊来源的细胞如何在某些微环境中生存的理解，例如口腔粘膜。新的方面是我们期望嗅鞘细胞对利基的形成和有助于细胞生存的基因表达谱的调节做出贡献。

Overall program goal The overall goal of this research program is to unravel the mechanisms that regulate the niche and function of oral/gingival neural crest derived stromal stem cells (NCSSCs). The NCSSCs are retained in a few specific locationssuch as bone marrow, hair follicles and oral mucosain adult tissue and their numbers generally decline with age. Using Wnt1-CRE/R26RYFP reporter mice, we have shown using tracing experiments that gingival NCSSCs are enriched in the gingival connective tissue. Interestingly, the NCSSCs are enriched at the connective tissue papilla and more specifically in close proximity to gingival oral epithelial cells (OECs). It is well known that connective tissue and epithelial cells engage in molecular crosstalk that can regulate the functions of both cell types. The crosstalk between OECs and NCSSCs is, therefore, likely to be involved in the regulation of NCSSC niche and function. There is no information available in the literature on how OECs could regulate NCSSC niche or fate. In the present proposal, we are proposing a new paradigm that OECs release extracellular vesicles that participate in the regulation of NCSSC fate. General hypothesis The general hypothesis is that signals derived from adjacent OECs regulate the fate and niche of NCSSCs in adult gingival tissue. Because the signals need to be specific and protected from off-target effects, we further hypothesize that the OECNCSSC crosstalk is regulated by extracellular vesicles (EVs). The EVs, including exosomes (EXO) and microvesicles (MVs), provide safety and receptor-mediated specificity to this regulation. Objectives and methodology The objective of the proposal is to find out how OEC-derived EXOs and MVs regulate the function and expression of differentiation-related genes and proteins in NCSSCs. Furthermore, we will investigate how these vesicles regulate the specific differentiation pathways, including osteogenic, adipogenic, chondrogenic and neuronal lineages. We will use a NCSSC cell line (named Ginestin, with over 90% nestin-positive cells) that we have isolated from adult human gingiva as our target cell line, expose it to OEC vesicles and measure the output with RT-qPCR, Western blotting, proteomics, immunostainings and other microscopic techniques. Expected outcomes and significance This research program is expected to significantly increase our understanding of how neural crest derived cells can survive in certain microenvironments such as oral mucosa. The novel aspect is the way we expect the OECs to contribute to the formation of the niche and regulation of the gene expression profile that is conductive for cell survival.