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

项目摘要/摘要 颅面部骨骼组织由多种功能单位组成，既包括矿化的，也包括矿化的 非矿化成分。非矿化组织，如缝线、颅底联合软骨和 牙周韧带存在于矿化组织之间，在颅面生长发育和修复中起重要作用。 通过为出生后生命中的组织特异性干细胞提供一个合适的位置进行再生。当前的基于细胞的疗法 不能有效地重建干细胞的利基环境；因此，破坏性骨骼状况的恢复，如 与牙周病相关的颅面畸形和晚期牙槽骨丢失尚未得到治疗 到目前为止已经成为可能。颅面骨组织的功能性再生需要一种创新的方法 重建固有的干细胞及其支持的壁龛。在这项提案中，我们的目标是定义分子和 颅面骨谱发育可塑性的细胞学机制及其机制探讨 应用这些机制来增强内源再生能力的可能性。我们假设 出生后颅面骨骼细胞谱系的功能专用细胞可重建组织特异性 干细胞及其支持的壁龛通过谱系可塑性。我们将使用一个 体内克隆谱系追踪与单细胞和空间转录相结合的方法解开 与干细胞及其干细胞生态位的形成有关的基本分子和细胞事件。 我们将重点研究两种模型的颅底联合软骨和牙周组织。 发育中的头面部骨骼谱系可塑性。在目标1中，我们将描述Runx2的可塑性 软骨膜细胞在建立颅底联合软骨龛中的作用。我们假设Runx2是软骨膜 成纤维细胞通过发育在出生后的软骨中产生干细胞及其微环境 可塑性。我们将结合细胞谱系追踪实验和单细胞转录分析， 使用特征条码技术的高分辨率空间转录分析和CRISPR屏幕 明确Runx2发育可塑性和干细胞生成潜能的分子机制 出生后合并软骨的软骨膜细胞。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 成人牙根表面的成牙骨质细胞保持牙囊(DF)细胞样状态，并可在实验中 转化为牙根间充质祖细胞。我们将使用细胞谱系追踪的组合 实验，单细胞和大量转录和表观基因组分析来定义PTHrP如何 成牙骨质细胞与PTHrP DF细胞相关，并在牙周过程中改变其分子特性 毁灭和再生。我们还将检查PTHrP的过度表达是否足以逆转 成熟的骨骼细胞在生长后阶段达到间充质祖细胞样状态，作为原则证明 测试发育谱系可塑性对成人阶段适用性的研究。

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.