• 牙卵泡：牙根形成和再生的中央调节剂

  牙卵泡：牙根形成和再生的中央调节剂

  牙根是牙齿的关键成分，通过牙周韧带（PDL）固定在周围的牙槽骨上。根系及其周围结构的适当形成不仅对于在营养摄入中咀嚼的基本功能，而且对于下部面部的生长和发育至关重要。普遍的牙齿疾病（例如龋齿和牙周疾病）是牙齿脱落的病因，只能通过缺乏功能结构的假体来治疗牙齿脱落，从而导致长期预后受损。需要一种有效的方法来再生功能牙周附着设备，以实现牙齿再生疗法的突破。在牙根形成期间，牙齿卵泡（DF）为牙骨质细胞，牙周韧带（PDL）细胞和肺泡隐性骨成骨细胞提供了前体细胞，以建立功能性牙周附着装置，即牙周。当前，牙科根部充质祖细胞的不同种群如何共同起作用并产生高功能性的牙根。
  
  在这个项目中，我们将定义如何在适当形成和再生牙齿根伴随功能性牙周附着装置的牙齿根中至关重要的不同类别的牙根间充质祖细胞的协同作用是至关重要的。
  
  在AIM 1中，我们将定义刺猬福克斯途径如何调节DF间充质祖细胞。我们假设叉子（FOX）转录因子在调节刺猬反应性DF间充质祖细胞的生理功能方面起着关键作用。我们将首先使用单细胞RNA-SEQ分析揭示刺猬反应性GLI1+ DF细胞的多样性，并确定它们与PTHRP+ DF细胞和其他前体细胞群的关系。其次，我们将使用诱导的细胞消融实验来确定Gli1+，PTHRP+和Runx2+细胞在牙周形成中的功能，并进一步测试FOXF1和FOXF2在牙周形成中使用floxed等位基因的功能。
  
  在目标2,我们将确定Wnt-mediated角色的pical root mesenchymal progenitor cells in tooth root formation. We hypothesize that chemokine (C-X-C motif) ligand 12 (CXCL12)+ mesenchymal progenitor cells in the apical root area orchestrate formation of the tooth root and the apical periodontium in a canonical Wnt signaling-mediated manner. We will determine the relationship between CXCL12+ cells and stem cells for apical papilla (SCAP) using ex vivo culture system, and define molecular mechanisms underlying a Wnt-mediated cell fate choice of mesenchymal progenitor cells by a comparative RNA-seq analysis followed by in situ validation. In Aim 3, we will determine actions of dental root mesenchymal progenitor cells in periodontal regeneration. We hypothesize that distinct classes of dental root mesenchymal progenitor cells contribute to regeneration in a concerted manner. We will define how descendants of Gli1+, PTHrP+, Runx2+ and CXCL12+ progenitor cells contribute to periodontal regeneration after bone destruction, by utilizing a ligature-induced periodontitis model mimicking periodontal diseases, and a surgical periodontal defect model.
  
  资金：R01DE029181

• Pharmacological rescue of tooth eruption disorders

  Pharmacological rescue of tooth eruption disorders

  A healthy dentition with appropriately functioning teeth is essential for maintaining the quality of life. Disorders involving tooth eruption are prevalent, as high as 2–4% in permanent molars alone. Multiple molars are involved in severe cases such as rare genetic conditions of primary failure of tooth eruption (PFE) or, more commonly, bisphosphonate-induced arrest of tooth eruption in pediatric patients, which significantly compromise the patients’ ability to chew effectively. Innovative adjuvant therapies for conventional orthodontic approaches are needed to facilitate tooth eruption of affected molars for better clinical outcomes. Tooth eruption is regulated by cells in the dental follicle (DF) surrounding developing molars that express parathyroid hormone-related protein (PTHrP, thereafter PTHrP+ DF cells). Inactivation of PTH/PTHrP receptor (PTH1R) in PTHrP+ DF cells causes failure of tooth eruption in murine molars that closely recapitulates the human PFE condition.
  
  In this project, we aim to develop pharamological modalities to rescue tooth eruption disorder. We hypothesize that activation of the PTH1R signaling pathway can restore defective tooth eruption of molars in mouse models of genetically and pharmacologically-induced tooth eruption disorders.
  

• 软骨肉瘤开始中的干细胞相互作用

  软骨肉瘤开始中的干细胞相互作用

  我们正在开发一种小鼠伦敦的小鼠模型，这是一种恶性中央软骨肉瘤的前体病变。软骨肿瘤是骨骼中最常见的原发性肿瘤，高度软骨肉瘤众所周知，对常规的非侵入性疗法具有侵略性且高度抗性。膜状瘤通常是由生命的头十年中产生的，具有肿瘤内镶嵌性的共同特征。在狭窄的发育窗口中，这些软骨肿瘤如何从少量突变细胞中发展出来。顿代二裂是在产后出现的，以促进生长板静息区（RZ）中特殊的软骨软骨细胞的特殊子集的自我更新。我们假设突变骨骼干细胞编排了软骨肿瘤的形成。
  
  在这个项目中，我们旨在了解表达PTHRP的骨骼干细胞与其壁s细胞之间在产后生长板的静息区之间的细胞间相互作用，并确定异常界面间相互作用如何导致软骨肿瘤的形成。

• 颅面骨骼细胞谱系可塑性，用于重建干细胞及其壁ni

  颅面骨骼细胞谱系可塑性，用于重建干细胞及其壁ni

  颅面骨骼组织由多个功能单元组成，包括矿化和非矿化成分。非矿物化组织，例如缝合线，颅底同步性和牙周韧带在矿化组织之间存在，并通过为颅面生长和再生起着重要作用，通过为后期生命中的组织特异性干细胞提供利基。当前基于细胞的疗法无法有效地重构干细胞壁ni。结果，迄今为止尚未使毁灭性的骨骼状况（例如颅面畸形和与牙周疾病相关的晚期肺泡骨质流失）恢复。颅面骨骼组织的功能再生需要一种创新的方法来重新建立固有的干细胞及其支撑壁球。在此提案中，我们旨在定义颅面骨骼谱系发育可塑性的分子和细胞机制，并探索应用这些机制以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通过谱系可塑性来重建组织特异性干细胞及其支持的小甲基细胞。
  
  我们将使用体内克隆谱系追踪以及单细胞和空间转录组方法的结合来检验这一假设，以揭开与干细胞及其干细胞生态裂市场相关的基本分子和细胞事件。我们将专注于两个颅底同步症的模型和牙周研究，以研究发育性颅面骨骼谱系可塑性。
  
  在AIM 1中，我们将表征Runx2+周围细胞的可塑性，以建立颅底同步性生殖位。我们假设runx2+ perongondrial成纤维细胞通过发育可塑性在产后同步性内产生干细胞及其壁ni。我们将结合使用特征条形码技术来定义发育可塑性和runx2+ perichondial runx2+ perichondial riper的潜在的分子机制，将细胞限制追踪实验和单细胞转录组，高分辨率空间转录组分析和CRISPR筛选结合使用。产后同步症。
  
  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.
  
  资金：R01DE030630

• Dynamics and Regulations of Bone Stem Cells In Vivo

  Dynamics and Regulations of Bone Stem Cells In Vivo

  儿童和年轻人普遍存在骨骼疾病和畸形。由于缺乏再生生长骨骼的有效方式，这些年轻患者经常接受多种手术干预措施，对他们，他们的家人和社会造成了重大负担。在骨骼生长过程中，软骨细胞和成骨细胞会连续产生，以使骨骼更大，更强壮。尚未完全理解用作这些细胞来源的内源性骨干细胞。关于这些骨干细胞如何协调内软骨和膜内骨化过程的基本知识对于开发可靠的生长骨骼的可靠方法至关重要。
  
  在这个项目中，将确定不同类型的骨干细胞的特征，这些特征将确定积极促进骨骼生长的特征。我们假设出生后生长板中的一部分静息软骨细胞以生长相关的骨干细胞的形式行为，并成为骨髓中间充质基质祖细胞的来源。这两种类型的骨干/祖细胞共同促进了适当的骨骼生长和维持。识别骨干细胞的特征和分子法规将促进我们通过再生工程来繁殖这些细胞的努力。
  
  In Aim1, we will identify molecular mechanisms regulating properties and fates of resting chondrocytes. The working hypothesis is that resting chondrocytes of the postnatal growth plate exhibit unique characteristics as growth-associated bone stem cells, whose properties and fates are regulated by Hedgehog signaling. We will first identify a self-renewing multipotent subpopulation of resting chondrocytes using in vitro colony-forming assays and in vivo transplantation of isolated growth plate cells. We will second identify the unique gene expression profiles of self-renewing colony-forming resting chondrocytes. We will further define roles of Hedgehog signaling in determining self-renewal and differentiation of resting chondrocytes by modulating its signaling components, while simultaneously tracing their behavior both in vivo and in vitro.
  
  In Aim2, we will define formation and fates of bone marrow mesenchymal stromal progenitors in growing bones. The working hypothesis is that growth plate chondrocytes undergo hypertrophy and transform into Cxcl12-abundant reticular (CAR) cells in bone marrow that behave as regional and reactive mesenchymal stromal progenitor cells. We will first define differentiation potentials of CAR cells into osteoblasts and adipocytes in vivo through intermittent PTH administration and a high-fat diet containing rosiglitazone. We will second determine CAR cells’ response to injury using a semistabilized tibial fracture model. We will also identify effects of these manipulations on CAR cells’ expression levels of key transcription factors that regulate cell fate choice. We will third define the properties of CAR cells as mesenchymal stromal progenitors through in vitro colony-forming assays and in vivo transplantation of isolated bone marrow cells. We will further define roles of canonical Wnt signaling as a cell fate determinant of osteoblast versus adipocyte differentiation using its floxed allele.
  
  资金：R01DE026666

主要出版物

1. Matsushita Y et al. The fate of early perichondrial cells in developing bones. Nat Commun. 2022. 13(1):7319.

2. Hallett SA等。生长板的静息区中的软骨细胞保持在WNT抑制环境中。Elife。2021. 10：E64513。

3. Matsushita Y等。WNT介导的骨髓基质细胞身份的转化支持骨骼再生。纳特社区。2020. 11（1）：332。

4.高桥A等。间充质祖细胞命运的自分泌调节策划牙齿喷发。Proc Natl Acad Sci U SA.2019。116（2）：575-580。

5. Mizuhashi K et al. Resting zone houses a unique class of skeletal stem cells. Nature. 2018. 563(7730):254-258.