Growth Plate Borderline Chondrocytes Behave as Transient Mesenchymal Precursor Cells

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly at the periphery of the growth plate immediately adjacent to the perichondrium are “borderline” chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single‐cell RNA‐seq analysis revealed subpopulations of Col2a1‐creER‐marked neonatal chondrocytes and their cell type–specific markers. A tamoxifen pulse to Pthrp‐creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow, and became Col(2.3 kb)‐GFP+ osteoblasts and Cxcl12‐GFPhigh reticular stromal “CAR” cells. Interestingly, these borderline chondrocyte‐derived marrow cells were short‐lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage‐tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. © 2019 American Society for Bone and Mineral Research.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/1/jbmr3719_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/2/jbmr3719-sup-0001-Suppl_Info_JBMR_021819.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151266/3/jbmr3719.pd

A Subset of Chondrogenic Cells Provides Early Mesenchymal Progenitors in Growing Bones

The hallmark of endochondral bone development is the presence of cartilaginous templates, in which osteoblasts and stromal cells are generated to form mineralized matrix and support bone marrow hematopoiesis. However, the ultimate source of these mesenchymal cells and the relationship between bone progenitors in fetal life and those in later life are unknown. Fate-mapping studies revealed that cells expressing cre-recombinases driven by the collagen II (Col2) promoter/enhancer and their descendants contributed to, in addition to chondrocytes, early perichondrial precursors prior to Runx2 expression and, subsequently, to a majority of osteoblasts, Cxcl12 (chemokine (C-X-C motif) ligand 12)-abundant stromal cells and bone marrow stromal/mesenchymal progenitor cells in postnatal life. Lineage-tracing experiments using a tamoxifen-inducible creER system further revealed that early postnatal cells marked by Col2-creER, as well as Sox9-creER and aggrecan (Acan)-creER, progressively contributed to multiple mesenchymal lineages and continued to provide descendants for over a year. These cells are distinct from adult mesenchymal progenitors and thus provide opportunities for regulating the explosive growth that occurs uniquely in growing mammals

A three‐dimensional analysis of primary failure of eruption in humans and mice

ObjectivesPrimary failure of eruption (PFE) is a genetic disorder exhibiting the cessation of tooth eruption. Loss‐of‐function mutations in parathyroid hormone (PTH)/parathyroid hormone‐related peptide (PTHrP) receptor (PTH/PTHrP receptor, PPR) were reported as the underlying cause of this disorder in humans. We showed in a PFE mouse model that PTHrP‐PPR signaling is responsible for normal dental follicle cell differentiation and tooth eruption. However, the mechanism underlying the eruption defect in PFE remains undefined. In this descriptive study, we aim to chronologically observe tooth eruption and root formation of mouse PFE molars through 3D microCT analyses.Setting and Sample PopulationTwo individuals with PFE were recruited at Showa University. A mouse PFE model was generated by deleting PPR specifically in PTHrP‐expressing dental follicle and divided into three groups, PPRfl/fl;R26RtdTomato/+(Control), PTHrP‐creER;PPRfl/+;R26RtdTomato/+(cHet), and PTHrP‐creER;PRRfl/fl;R26RtdTomato/+(cKO).Materials and MethodsImages from human PFE subjects were acquired by CBCT. All groups of mouse samples were studied at postnatal days 14, 25, 91, and 182 after a tamoxifen pulse at P3, and superimposition of 3D microCT images among three groups was rendered.ResultsMouse and human PFE molars exhibited a similar presentation in the 3D CT analyses. The quantitative analysis in mice demonstrated a statistically significant decrease in the eruption height of cKO first and second molars compared to other groups after postnatal day 25. Additionally, cKO molars demonstrated significantly shortened roots with dilacerations associated with the reduced interradicular bone height.ConclusionsMouse PFE molars erupt at a much slower rate compared to normal molars, associated with shortened and dilacerated roots and defective interradicular bones.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154523/1/odi13249_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154523/2/odi13249.pd

Bone regeneration via skeletal cell lineage plasticity: All hands mobilized for emergencies

An emerging concept is that quiescent mature skeletal cells provide an important cellular source for bone regeneration. It has long been considered that a small number of resident skeletal stem cells are solely responsible for the remarkable regenerative capacity of adult bones. However, recent in vivo lineage‐tracing studies suggest that all stages of skeletal lineage cells, including dormant pre‐adipocyte‐like stromal cells in the marrow, osteoblast precursor cells on the bone surface and other stem and progenitor cells, are concomitantly recruited to the injury site and collectively participate in regeneration of the damaged skeletal structure. Lineage plasticity appears to play an important role in this process, by which mature skeletal cells can transform their identities into skeletal stem cell‐like cells in response to injury. These highly malleable, long‐living mature skeletal cells, readily available throughout postnatal life, might represent an ideal cellular resource that can be exploited for regenerative medicine.An emerging concept is that quiescent mature skeletal cells provide important cellular sources for bone regeneration though lineage plasticity, by which these cells transform their identities into skeletal stem cell‐like cells in response to injury. These long‐living mature skeletal cells available throughout adult life might be exploited for regenerative medicine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/163968/1/bies202000202_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/163968/2/bies202000202.pd

The hypertrophic chondrocyte: To be or not to be

Hypertrophic chondrocytes are the master regulators of endochondral ossification; however, their ultimate cell fates cells remain largely elusive due to their transient nature. Historically, hypertrophic chondrocytes have been considered as the terminal state of growth plate chondrocytes, which are destined to meet their inevitable demise at the primary spongiosa. Chondrocyte hypertrophy is accompanied by increased organelle synthesis and rapid intracellular water uptake, which serve as the major drivers of longitudinal bone growth. This process is delicately regulated by major signaling pathways and their target genes, including growth hormone (GH), insulin growth factor-1 (IGF-1), indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), bone morphogenetic proteins (BMPs), sex determining region Y-box 9 (Sox9), runt-related transcription factors (Runx) and fibroblast growth factor receptors (FGFRs). Hypertrophic chondrocytes orchestrate endochondral ossification by regulating osteogenic-angiogenic and osteogenic-osteoclastic coupling through the production of vascular endothelial growth factor (VEGF), receptor activator of nuclear factor kappa-B ligand (RANKL) and matrix metallopeptidases-9/13 (MMP-9/13). Hypertrophic chondrocytes also indirectly regulate resorption of the cartilaginous extracellular matrix, by controlling formation of a special subtype of osteoclasts termed "chondroclasts". Notably, hypertrophic chondrocytes may possess innate potential for plasticity, reentering the cell cycle and differentiating into osteoblasts and other types of mesenchymal cells in the marrow space. We may be able to harness this unique plasticity for therapeutic purposes, for a variety of skeletal abnormalities and injuries. In this review, we discuss the morphological and molecular properties of hypertrophic chondrocytes, which carry out important functions during skeletal growth and regeneration

Multi-omics analysis in developmental bone biology

Single-cell omics and multi-omics have revolutionized our understanding of molecular and cellular biological processes at a single-cell level. In bone biology, the combination of single-cell RNA-sequencing analyses and in vivo lineage-tracing approaches has successfully identified multi-cellular diversity and dynamics of skeletal cells. This established a new concept that bone growth and regeneration are regulated by concerted actions of multiple types of skeletal stem cells, which reside in spatiotemporally distinct niches. One important subtype is endosteal stem cells that are particularly abundant in young bone marrow. The discovery of this new skeletal stem cell type has been facilitated by single-cell multi-omics, which simultaneously measures gene expression and chromatin accessibility. Using single-cell omics, it is now possible to computationally predict the immediate future state of individual cells and their differentiation potential. In vivo validation using histological approaches is the key to interpret the computational prediction. The emerging spatial omics, such as spatial transcriptomics and epigenomics, have major advantage in retaining the location of individual cells within highly complex tissue architecture. Spatial omics can be integrated with other omics to further obtain in-depth insights. Single-cell multi-omics are now becoming an essential tool to unravel intricate multicellular dynamics and intercellular interactions of skeletal cells

Parathyroid hormone receptor signalling in osterix-expressing mesenchymal progenitors is essential for tooth root formation

Dental root formation is a dynamic process in which mesenchymal cells migrate toward the site of the future root, differentiate and secrete dentin and cementum. However, the identities of dental mesenchymal progenitors are largely unknown. Here we show that cells expressing osterix are mesenchymal progenitors contributing to all relevant cell types during morphogenesis. The majority of cells expressing parathyroid hormone-related peptide (PTHrP) are in the dental follicle and on the root surface, and deletion of its receptor (PPR) in these progenitors leads to failure of eruption and significantly truncated roots lacking periodontal ligaments. The PPR-deficient progenitors exhibit accelerated cementoblast differentiation with upregulation of nuclear factor I/C (Nfic). Deletion of histone deacetylase-4 (HDAC4) partially recapitulates the PPR deletion root phenotype. These findings indicate that PPR signalling in dental mesenchymal progenitors is essential for tooth root formation, underscoring importance of the PTHrP–PPR system during root morphogenesis and tooth eruption

Intercellular Interactions of an Adipogenic CXCL12‐Expressing Stromal Cell Subset in Murine Bone Marrow

Bone marrow houses a multifunctional stromal cell population expressing C‐X‐C motif chemokine ligand 12 (CXCL12), termed CXCL12‐abundant reticular (CAR) cells, that regulates osteogenesis and adipogenesis. The quiescent pre‐adipocyte‐like subset of CXCL12+ stromal cells (“Adipo‐CAR” cells) is localized to sinusoidal surfaces and particularly enriched for hematopoiesis‐supporting cytokines. However, detailed characteristics of these CXCL12+ pre‐adipocyte‐like stromal cells and how they contribute to marrow adipogenesis remain largely unknown. Here we highlight CXCL12‐dependent physical coupling with hematopoietic cells as a potential mechanism regulating the adipogenic potential of CXCL12+ stromal cells. Single‐cell computational analyses of RNA velocity and cell signaling reveal that Adipo‐CAR cells exuberantly communicate with hematopoietic cells through CXCL12‐CXCR4 ligand‐receptor interactions but do not interconvert with Osteo‐CAR cells. Consistent with this computational prediction, a substantial fraction of Cxcl12‐creER+ pre‐adipocyte‐like cells intertwines with hematopoietic cells in vivo and in single‐cell preparation in a protease‐sensitive manner. Deletion of CXCL12 in these cells using Col2a1‐cre leads to a reduction of stromal‐hematopoietic coupling and extensive marrow adipogenesis in adult bone marrow, which appears to involve direct conversion of CXCL12+ cells to lipid‐laden marrow adipocytes without altering mesenchymal progenitor cell fates. Therefore, these findings suggest that CXCL12+ pre‐adipocyte‐like marrow stromal cells prevent their premature differentiation by maintaining physical coupling with hematopoietic cells in a CXCL12‐dependent manner, highlighting a possible cell‐non‐autonomous mechanism that regulates marrow adipogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/168264/1/jbmr4282.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/168264/2/jbmr4282_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/168264/3/jbmr4282-sup-0001-Supinfo.pd