• Dental Follicle: A Central Regulator of Tooth Root Formation and Regeneration

  Dental Follicle: A Central Regulator of Tooth Root Formation and Regeneration

  The tooth root is a critical component of the tooth, anchored to surrounding alveolar bones through the periodontal ligament (PDL). Appropriate formation of the root and its surrounding structure is essential not only for fundamental functions of the tooth in mastication for nutrition intake, but also for growth and development of the lower face. Prevalent dental diseases such as caries and periodontal diseases are the etiology for tooth loss, which can be treated only by prostheses lacking functional structures, leading to compromised long-term prognosis. An effective approach to regenerate a functional periodontal attachment apparatus is needed to achieve a breakthrough in dental regenerative therapies. During tooth root formation, the dental follicle (DF) provides precursor cells for cementoblasts, periodontal ligament (PDL) cells and alveolar cryptal bone osteoblasts to establish the functional periodontal attachment apparatus, the periodontium. Currently, how diverse populations of dental root mesenchymal progenitor cells work together and generate highly functional tooth roots remains unknown.
  
  In this project, we will define how concerted actions of distinct classes of dental root mesenchymal progenitor cells are essential for proper formation and regeneration of the tooth root accompanied by a functional periodontal attachment apparatus.
  
  In Aim 1, we will define how Hedgehog-Fox pathway regulates DF mesenchymal progenitor cells. We hypothesize that the Forkhead (Fox) transcription factors play a key role in regulating physiological functions of Hedgehog-responsive DF mesenchymal progenitor cells. We will first reveal the diversity of Hedgehog-responsive Gli1+ DF cells using single cell RNA-seq analyses, and determine their relationships with PTHrP+ DF cells and other precursor cell populations. Second, we will determine the function of Gli1+, PTHrP+ and Runx2+ cells in periodontium formation using inducible cell ablation experiments, and further test the function of Foxf1 and Foxf2 in periodontium formation using their floxed alleles.
  
  In Aim 2, we will identify Wnt-mediated roles of apical 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.
  
  Funding: 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.
  

• Stem cell interactions in chondrosarcoma initiation

  Stem cell interactions in chondrosarcoma initiation

  We are developing a mouse model of enchondroma, a precursor lesion for malignant central chondrosarcoma. Cartilage tumors are the most common primary neoplasms in bones, and high-grade chondrosarcoma is notoriously aggressive and highly resistant to conventional non-invasive therapies. Enchondroma typically arises from the growth plate in the first decade of life with common characteristics of intra-neoplastic mosaicism. How these cartilage tumors develop from a small number of mutant cells during a narrow developmental window remains undefined. The epiphyseal niche develops postnatally to facilitate self-renewal of a special subset of cartilage-forming chondrocytes in the resting zone (RZ) of the growth plate. We hypothesize that mutant skeletal stem cells orchestrate cartilage tumor formation.
  
  In this project, we aim to understand intercellular interactions between PTHrP-expressing skeletal stem cells and their niche cells within the resting zone of the postnatal growth plate, and define how aberrant intercelluar interactions may lead to cartilage tumor formation.

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

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

  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.
  
  Funding: R01DE030630

• Dynamics and Regulations of Bone Stem Cells In Vivo

  Dynamics and Regulations of Bone Stem Cells In Vivo

  Bone disorders and deformities are prevalent in children and young adults. Due to lack of effective modalities to regenerate growing bones, these young patients often undergo multiple surgical interventions, posing a significant burden on them, their family and society. During bone growth, chondrocytes and osteoblasts are continuously generated to make bones bigger and stronger. Endogenous bone stem cells that serve as the source of these cells have not been completely understood. Fundamental knowledge of how these bone stem cells coordinate the two processes of endochondral and intramembranous ossification is essential to develop a reliable approach to regenerate growing bones.
  
  In this project, the characteristics of distinct types of bone stem cells that actively promote bone growth will be identified. We hypothesize that a subset of resting chondrocytes in the postnatal growth plate behave as growth-associated bone stem cells, and become a source of mesenchymal stromal progenitor cells in bone marrow; these two types of bone stem/progenitor cells concertedly promote proper bone growth and maintenance. Identifying characteristics and molecular regulations of bone stem cells will facilitate our endeavor to reproduce these cells through regenerative engineering.
  
  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.
  
  Funding: R01DE026666

Key Publications

1. Matsushita Y et al. Bone marrow endosteal stem cells dictate active osteogenesis and aggressive tumorigenesis. Nat Commun. 2023. 14(1):2383.

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

3. Hallett SA et al. Chondrocytes in the resting zone of the growth plate are maintained in a Wnt-inhibitory environment. Elife. 2021. 10:e64513.

4. Matsushita Y et al. A Wnt-mediated conversion of the bone marrow stromal cell identity supports skeletal regeneration. Nat Commun. 2020. 11(1):332.

5. Takahashi A et al. Autocrine regulation of mesenchymal progenitor cell fates orchestrates tooth eruption. Proc Natl Acad Sci U S A. 2019. 116(2):575-580.

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