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

项目总体目标****本研究项目的总体目标是揭示口腔/牙龈神经嵴源性基质干细胞（NCSSCs）生态位和功能的调控机制。NCSSCs保留在一些特定的位置，如骨髓、毛囊和成人组织的口腔黏膜，它们的数量通常随着年龄的增长而下降。使用Wnt1-CRE/R26RYFP报告小鼠，我们通过示踪实验发现，牙龈NCSSCs在牙龈结缔组织中富集。有趣的是，NCSSCs在结缔组织乳头上富集，更具体地说，在牙龈口腔上皮细胞（OECs）附近富集。众所周知，结缔组织和上皮细胞参与分子串扰，可以调节两种细胞类型的功能。因此，oec和NCSSCs之间的串扰可能参与了NCSSC生态位和功能的调控。文献中没有关于OECs如何调节NCSSC生态位或命运的信息。在本文中，我们提出了一种新的范式，即oec释放参与NCSSC命运调控的细胞外囊泡。*******一般假设****一般假设是来自相邻oec的信号调节成人牙龈组织中NCSSCs的命运和生态位。由于信号需要特异性和免受脱靶效应的影响，我们进一步假设OECNCSSC串扰是由细胞外囊泡（ev）调节的。包括外泌体（EXO）和微囊泡（mv）在内的ev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****该提案的目的是找出oec衍生的exo和MVs如何调节NCSSCs中分化相关基因和蛋白质的功能和表达。此外，我们将研究这些囊泡如何调节特定的分化途径，包括成骨、脂肪、软骨和神经元谱系。我们将使用从成人牙龈中分离的NCSSC细胞系（命名为Ginestin，巢蛋白阳性细胞超过90%）作为我们的靶细胞系，将其暴露于OEC囊泡中，并使用RT-qPCR， Western blotting，蛋白质组学，免疫染色等显微技术测量输出。*******预期结果和意义****本研究计划有望显著增加我们对神经嵴来源细胞如何在某些微环境（如口腔粘膜）中存活的理解。新颖的方面是我们期望oec有助于形成有利于细胞存活的生态位和调控基因表达谱的方式。******

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.******