Craniofacial skeletal cell lineage plasticity for reconstituting stem cells and their niches

项目总结/摘要 颅面骨骼组织由多个功能单位组成，包括矿化和 非矿化成分。非矿化组织，如骨缝、颅底软骨结合和 牙周韧带，存在于矿化组织之间，在颅面生长中起重要作用， 通过为出生后的组织特异性干细胞提供一个小生境来促进再生。当前基于细胞的疗法 不能有效地重建干细胞龛;因此，恢复破坏性的骨骼条件， 与牙周病相关的颅面畸形和晚期牙槽骨丢失尚未被 使之成为可能。颅面骨骼组织的功能性再生需要创新的方法 来重建固有的干细胞及其支持生态位。在这个建议中，我们的目标是定义分子和 颅面骨骼谱系发育可塑性的细胞机制，并探讨 应用这些机制来增强内源性再生能力的可能性。我们假设 出生后颅面骨骼细胞谱系的功能专用细胞可以重建组织特异性 干细胞及其支持生态位通过谱系可塑性。我们将使用一个 结合体内克隆谱系追踪和单细胞及空间转录组学方法， 与干细胞及其干细胞生态位形成相关的基本分子和细胞事件。 我们将重点研究颅底软骨结合和牙周组织两种模型 发育性颅面骨骼谱系可塑性在目标1中，我们将描述Runx 2+的可塑性， 软骨膜细胞在建立颅底软骨结合龛中的作用。我们假设Runx2+软骨膜 成纤维细胞通过发育过程在出生后的软骨结合中产生干细胞和它们的小生境 可塑性我们将结合细胞谱系追踪实验和单细胞转录组学分析， 高分辨率空间转录组学分析和CRISPR筛选，使用特征条形码技术， 确定Runx2+的发育可塑性和干细胞生成潜力的分子机制 出生后软骨结合的软骨膜细胞。在目标2中，我们将探索重新激活PTHrP+的可能性。 成牙骨质细胞再生功能性牙周附着装置。我们假设PTHrP + 成年牙根表面上的成牙骨质细胞保持牙囊（DF）细胞样状态，并且可以实验性地 恢复为牙根间充质祖细胞。我们将结合细胞谱系追踪 实验，单细胞和批量转录组和表观基因组分析，以确定PTHrP + 成牙骨质细胞与PTHrP + DF细胞相关，并在牙周炎期间改变其分子身份， 毁灭与再生我们还将研究PTHrP过表达是否足以逆转 在生长后阶段将成熟骨骼细胞转化为间充质祖细胞样状态，作为原理证明 一项旨在测试发育谱系可塑性对成年阶段的适用性的研究。

PROJECT SUMMARY/ABSTRACT The craniofacial skeletal tissues are composed of multiple functional units, encompassing both mineralized and non-mineralized components. The non-mineralized tissues, such as sutures, cranial base synchondroses and periodontal ligaments, exist between mineralized tissues, and play important roles in craniofacial growth and regeneration by providing a niche for tissue-specific stem cells in postnatal life. Current cell-based therapies cannot effectively reconstitute stem cell niches; as a result, recovery of devastating skeletal conditions such as craniofacial deformities and advanced alveolar bone loss associated with periodontal diseases has not been made possible to date. Functional regeneration of craniofacial skeletal tissues requires an innovative approach to reestablish inherent stem cells and their supporting niches. In this proposal, we aim to define molecular and cellular mechanisms underlying developmental plasticity of the craniofacial skeletal lineage and explore the possibility to apply these mechanisms to enhance endogenous regeneration capacity. We hypothesize that functionally dedicated cells of the postnatal craniofacial skeletal cell lineage can reconstitute tissue-specific stem cells and their supporting niches through lineage plasticity. We will test this hypothesis using a combination of in vivo clonal lineage-tracing and single-cell and spatial transcriptomic approaches to unravel fundamental molecular and cellular events associated with formation of stem cells and their stem cell niche. We will focus on two models of the cranial base synchondrosis and the periodontium to investigate developmental craniofacial skeletal lineage plasticity. In Aim 1, we will characterize plasticity of Runx2+ perichondrial cells in establishing the cranial base synchondrosis niche. We hypothesize Runx2+ perichondrial fibroblasts generate both stem cells and their niches within postnatal synchondroses through developmental plasticity. We will use a combination of cell-lineage tracing experiments and single-cell transcriptomic analyses, high-resolution spatial transcriptomic analysis and CRISPR screens using the feature barcoding technology to define molecular mechanisms underlying developmental plasticity and stem cell-generating potential of Runx2+ perichondrial cells of the postnatal synchondrosis. In Aim 2, we will explore the possibility to reactivate PTHrP+ cementoblasts to regenerate functional periodontal attachment apparatus. We hypothesize that PTHrP+ cementoblasts on the adult root surface retain a dental follicle (DF) cell-like state, and can be experimentally reverted to dental root mesenchymal progenitor cells. We will use a combination of cell-lineage tracing experiments, single-cell and bulk transcriptomic and epigenomic analyses to define how PTHrP+ cementoblasts are related to PTHrP+ DF cells, and change their molecular identities during periodontal destruction and regeneration. We will also examine whether PTHrP overexpression is sufficient to revert mature skeletal cells to a mesenchymal progenitor-like state at a post-growth phase, as a proof-of-principle study to test the applicability of developmental lineage plasticity to adult stages.