ABSTRACT
Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.
Subject(s)
Bone Development/physiology , Bone and Bones/cytology , Hematopoietic Stem Cells/cytology , Animals , Bone and Bones/metabolism , Cartilage/cytology , Cell Differentiation , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C57BL , Signal Transduction , Single-Cell Analysis/methods , Stem Cells/cytology , Stromal Cells/cytology , Transcriptome/geneticsABSTRACT
How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.
Subject(s)
Bone and Bones/cytology , Mesenchymal Stem Cells/cytology , Animals , Bone Morphogenetic Proteins/metabolism , Cartilage/cytology , Cell Lineage , Crosses, Genetic , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Signal TransductionABSTRACT
The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).
Subject(s)
Bone and Bones/cytology , Intercellular Signaling Peptides and Proteins/metabolism , Intestine, Small/cytology , Mesenchymal Stem Cells/cytology , Animals , Cartilage/metabolism , Intestine, Small/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BLABSTRACT
Load-bearing tissues, such as muscle and cartilage, exhibit high elasticity, high toughness and fast recovery, but have different stiffness (with cartilage being significantly stiffer than muscle)1-8. Muscle achieves its toughness through finely controlled forced domain unfolding-refolding in the muscle protein titin, whereas articular cartilage achieves its high stiffness and toughness through an entangled network comprising collagen and proteoglycans. Advancements in protein mechanics and engineering have made it possible to engineer titin-mimetic elastomeric proteins and soft protein biomaterials thereof to mimic the passive elasticity of muscle9-11. However, it is more challenging to engineer highly stiff and tough protein biomaterials to mimic stiff tissues such as cartilage, or develop stiff synthetic matrices for cartilage stem and progenitor cell differentiation12. Here we report the use of chain entanglements to significantly stiffen protein-based hydrogels without compromising their toughness. By introducing chain entanglements13 into the hydrogel network made of folded elastomeric proteins, we are able to engineer highly stiff and tough protein hydrogels, which seamlessly combine mutually incompatible mechanical properties, including high stiffness, high toughness, fast recovery and ultrahigh compressive strength, effectively converting soft protein biomaterials into stiff and tough materials exhibiting mechanical properties close to those of cartilage. Our study provides a general route towards engineering protein-based, stiff and tough biomaterials, which will find applications in biomedical engineering, such as osteochondral defect repair, and material sciences and engineering.
Subject(s)
Biocompatible Materials , Cartilage , Hydrogels , Biocompatible Materials/chemical synthesis , Biocompatible Materials/chemistry , Cartilage/chemistry , Collagen/chemistry , Connectin/chemistry , Hydrogels/chemical synthesis , Hydrogels/chemistry , Proteoglycans/chemistry , Tissue Engineering/methods , HumansABSTRACT
The neurocranium is an integral part of the vertebrate head, itself a major evolutionary innovation1,2. However, its early history remains poorly understood, with great dissimilarity in form between the two living vertebrate groups: gnathostomes (jawed vertebrates) and cyclostomes (hagfishes and lampreys)2,3. The 100 Myr gap separating the Cambrian appearance of vertebrates4-6 from the earliest three-dimensionally preserved vertebrate neurocrania7 further obscures the origins of modern states. Here we use computed tomography to describe the cranial anatomy of an Ordovician stem-group gnathostome: Eriptychius americanus from the Harding Sandstone of Colorado, USA8. A fossilized head of Eriptychius preserves a symmetrical set of cartilages that we interpret as the preorbital neurocranium, enclosing the fronts of laterally placed orbits, terminally located mouth, olfactory bulbs and pineal organ. This suggests that, in the earliest gnathostomes, the neurocranium filled out the space between the dermal skeleton and brain, like in galeaspids, osteostracans and placoderms and unlike in cyclostomes2. However, these cartilages are not fused into a single neurocranial unit, suggesting that this is a derived gnathostome trait. Eriptychius fills a major temporal and phylogenetic gap in our understanding of the evolution of the gnathostome head, revealing a neurocranium with an anatomy unlike that of any previously described vertebrate.
Subject(s)
Fossils , Phylogeny , Skull , Vertebrates , Animals , Hagfishes/anatomy & histology , Imaging, Three-Dimensional , Lampreys/anatomy & histology , Mouth , Olfactory Bulb , Pineal Gland , Skull/anatomy & histology , Tomography Scanners, X-Ray Computed , Vertebrates/anatomy & histology , Vertebrates/classification , Colorado , Cartilage/anatomy & histologyABSTRACT
Insufficient intracellular anabolism is a crucial factor involved in many pathological processes in the body1,2. The anabolism of intracellular substances requires the consumption of sufficient intracellular energy and the production of reducing equivalents. ATP acts as an 'energy currency' for biological processes in cells3,4, and the reduced form of NADPH is a key electron donor that provides reducing power for anabolism5. Under pathological conditions, it is difficult to correct impaired anabolism and to increase insufficient levels of ATP and NADPH to optimum concentrations1,4,6-8. Here we develop an independent and controllable nanosized plant-derived photosynthetic system based on nanothylakoid units (NTUs). To enable cross-species applications, we use a specific mature cell membrane (the chondrocyte membrane (CM)) for camouflage encapsulation. As proof of concept, we demonstrate that these CM-NTUs enter chondrocytes through membrane fusion, avoid lysosome degradation and achieve rapid penetration. Moreover, the CM-NTUs increase intracellular ATP and NADPH levels in situ following exposure to light and improve anabolism in degenerated chondrocytes. They can also systemically correct energy imbalance and restore cellular metabolism to improve cartilage homeostasis and protect against pathological progression of osteoarthritis. Our therapeutic strategy for degenerative diseases is based on a natural photosynthetic system that can controllably enhance cell anabolism by independently providing key energy and metabolic carriers. This study also provides an enhanced understanding of the preparation and application of bioorganisms and composite biomaterials for the treatment of disease.
Subject(s)
Chondrocytes , Osteoarthritis , Photosynthesis , Plants , Humans , Adenosine Triphosphate/metabolism , Chondrocytes/metabolism , NADP/metabolism , Osteoarthritis/metabolism , Osteoarthritis/pathology , Osteoarthritis/therapy , Plants/metabolism , Cartilage/cytology , Cartilage/metabolism , Homeostasis , Energy Metabolism , Membrane FusionABSTRACT
During endochondral ossification, chondrocytes secrete a proteoglycan (PG)-rich extracellular matrix that can inhibit the process of cartilage maturation, including expression of Ihh and Col10a1. Because bone morphogenetic proteins (BMPs) can promote cartilage maturation, we hypothesized that cartilage PGs normally inhibit BMP signalling. Accordingly, BMP signalling was evaluated in chondrocytes of wild-type and PG mutant (fam20b-/-) zebrafish and inhibited with temporal control using the drug DMH1 or an inducible dominant-negative BMP receptor transgene (dnBMPR). Compared with wild type, phospho-Smad1/5/9, but not phospho-p38, was increased in fam20b-/- chondrocytes, but only after they secreted PGs. Phospho-Smad1/5/9 was decreased in DMH1-treated or dnBMPR-activated wild-type chondrocytes, and DMH1 also decreased phospho-p38 levels. ihha and col10a1a were decreased in DMH1-treated or dnBMPR-activated chondrocytes, and less perichondral bone formed. Finally, early ihha and col10a1a expression and early perichondral bone formation of fam20b mutants were rescued with DMH1 treatment or dnBMPR activation. Therefore, PG inhibition of canonical BMP-dependent cartilage maturation delays endochondral ossification, and these results offer hope for the development of growth factor therapies for skeletal defects of PG diseases.
Subject(s)
Osteogenesis , Proteoglycans , Animals , Osteogenesis/genetics , Proteoglycans/genetics , Proteoglycans/metabolism , Zebrafish/genetics , Cartilage/metabolism , Chondrocytes/metabolism , Bone Morphogenetic Proteins/metabolismABSTRACT
Endochondral ossification contributes to longitudinal skeletal growth. Osteoblasts, which are bone-forming cells, appear close to terminally differentiated hypertrophic chondrocytes during endochondral ossification. We established mice with conditional knockout (cKO) of Smad4, an essential co-activator for transforming growth factor ß family signaling. The mice showed a marked increase in bone volume in the metaphysis as a result of increased bone formation by osteoblasts, in which ß-catenin, an effector of canonical Wnt signaling, accumulated. We identified Wnt7b as a factor with increased expression in growth plate cartilage in Smad4 cKO mice. Wnt7b mRNA was expressed in differentiated chondrocytes and suppressed by BMP4 stimulation. Ablation of Wnt7b blunted the increase in bone in adult Smad4 cKO mice and reduced skeletal growth in juvenile mice. Overall, we conclude that Wnt7b is a crucial factor secreted from hypertrophic chondrocytes to initiate endochondral ossification. These results suggest that Smad4-dependent BMP signaling regulates the Wnt7b-ß-catenin axis during endochondral ossification.
Subject(s)
Chondrocytes , Osteogenesis , Animals , Mice , beta Catenin/metabolism , Bone and Bones , Cartilage/metabolism , Cell Differentiation/genetics , Chondrocytes/metabolism , Osteogenesis/genetics , Proto-Oncogene Proteins/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolismABSTRACT
Non-canonical/ß-catenin-independent Wnt signaling plays crucial roles in tissue/cell polarity in epithelia, but its functions have been less well studied in mesenchymal tissues, such as the skeleton. Mutations in non-canonical Wnt signaling pathway genes cause human skeletal diseases such as Robinow syndrome and Brachydactyly Type B1, which disrupt bone growth throughout the endochondral skeleton. Ror2 is one of several non-canonical Wnt receptor/co-receptors. Here, we show that ror2-/- mutant zebrafish have craniofacial skeletal defects, including disruptions of chondrocyte polarity. ror1-/- mutants appear to be phenotypically wild type, but loss of both ror1 and ror2 leads to more severe cartilage defects, indicating partial redundancy. Skeletal defects in ror1/2 double mutants resemble those of wnt5b-/- mutants, suggesting that Wnt5b is the primary Ror ligand in zebrafish. Surprisingly, the proline-rich domain of Ror2, but not its kinase domain, is required to rescue its function in mosaic transgenic experiments in ror2-/- mutants. These results suggest that endochondral bone defects in ROR-related human syndromes reflect defects in cartilage polarity and morphogenesis.
Subject(s)
Chondrocytes , Zebrafish , Animals , Bone and Bones/metabolism , Cartilage/metabolism , Cell Polarity/genetics , Chondrocytes/metabolism , Morphogenesis/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Receptors, Wnt/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolism , Wnt Signaling Pathway/genetics , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish ProteinsABSTRACT
Skeletal growth promoted by endochondral ossification is tightly coordinated by self-renewal and differentiation of chondrogenic progenitors. Emerging evidence has shown that multiple skeletal stem cells (SSCs) participate in cartilage formation. However, as yet, no study has reported the existence of common long-lasting chondrogenic progenitors in various types of cartilage. Here, we identify Gli1+ chondrogenic progenitors (Gli1+ CPs), which are distinct from PTHrP+ or FoxA2+ SSCs, are responsible for the lifelong generation of chondrocytes in the growth plate, vertebrae, ribs, and other cartilage. The absence of Gli1+ CPs leads to cartilage defects and dwarfishness phenotype in mice. Furthermore, we show that the BMP signal plays an important role in self-renewal and maintenance of Gli1+ CPs. Deletion of Bmpr1α triggers Gli1+ CPs quiescence exit and causes the exhaustion of Gli1+ CPs, consequently disrupting columnar cartilage. Collectively, our data demonstrate that Gli1+ CPs are common long-term chondrogenic progenitors in multiple types of cartilage and are essential to maintain cartilage homeostasis.
Subject(s)
Cartilage , Chondrogenesis , Animals , Mice , Zinc Finger Protein GLI1/genetics , Chondrogenesis/genetics , Chondrocytes , Osteogenesis , Cell DifferentiationABSTRACT
Chondrocytes and osteoblasts differentiated from induced pluripotent stem cells (iPSCs) will provide insights into skeletal development and genetic skeletal disorders and will generate cells for regenerative medicine applications. Here, we describe a method that directs iPSC-derived sclerotome to chondroprogenitors in 3D pellet culture then to articular chondrocytes or, alternatively, along the growth plate cartilage pathway to become hypertrophic chondrocytes that can transition to osteoblasts. Osteogenic organoids deposit and mineralize a collagen I extracellular matrix (ECM), mirroring in vivo endochondral bone formation. We have identified gene expression signatures at key developmental stages including chondrocyte maturation, hypertrophy, and transition to osteoblasts and show that this system can be used to model genetic cartilage and bone disorders.
Subject(s)
Cartilage , Induced Pluripotent Stem Cells , Humans , Cartilage/metabolism , Chondrocytes/metabolism , Cell Differentiation , Osteoblasts , Induced Pluripotent Stem Cells/metabolismABSTRACT
Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.
Subject(s)
Aging , Osteoporosis , Phosphoric Diester Hydrolases , Pyrophosphatases , Animals , Humans , Mice , Aging/genetics , Cartilage/metabolism , Luciferases , Mice, Knockout , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/genetics , Pyrophosphatases/metabolismABSTRACT
The objective of this study was to discuss the mechanism of artesunate (ART) in improving cartilage damage in osteoarthritis (OA) by regulating the expression levels of metastatic tumor antigen 1 (MTA1), lipoxin A4 (LXA4) and the downstream JAK2/STAT3 signaling pathway. The OA model in vitro was constructed by stimulating chondrocytes for 24 h with 10 ng/mL interleukin (IL)-1ß, and cell proliferation and apoptosis, expression levels of Aggrecan, MTA1, LXA4, MMP3, MMP13 and Collagen II, and inflammatory cytokines in the culture supernatants were examined. Histopathological changes, inflammatory response and chondrocyte apoptosis of the cartilage tissues of OA mice were performed. In vitro cell experiments, ART enhanced cell proliferation capacity, accompanied by decreased apoptosis rate, decreased expression of MMP-3 and MMP-13, elevated expression of Collagen II and Aggrecan, as well as reduced levels of IL-6 and TNF-α in the cell supernatant. ART also ameliorated IL-1ß-induced chondrocyte damage by upregulating MTA1. The LXA4 promoter region had two potential binding sites for MTA1. There was a positive correlation between MTA1 and LXA4. MTA1 enhanced the expression of LXA4 through transcription and blocked the activation of the JAK2/STAT3 signaling pathway. In vivo animal model experiments further showed that ART treatment alleviated cartilage tissue damage in OA model mice by upregulating MTA1. Our study demonstrates that ART improves the cartilage damage of OA by upregulating MTA1 expression and promoting the transcriptional activation of LXA4, and further blocking the JAK2/STAT3 signaling pathway.
Subject(s)
Neoplasms , Osteoarthritis , Mice , Animals , Artesunate/metabolism , Transcriptional Activation , Aggrecans/metabolism , Cartilage/metabolism , Chondrocytes/metabolism , Osteoarthritis/pathology , Signal Transduction , Collagen/metabolism , Neoplasms/pathology , Interleukin-1beta/metabolismABSTRACT
Like rheumatoid arthritis (RA) in humans, collagen-induced arthritis (CIA) in mice is associated with not only MHC class II genetic polymorphism but also, to some extent, with other loci including genes encoding Fc gamma receptors (FCGRs) and complement C5. In this study, we used a cartilage antibody-induced arthritis (CAIA) model in which arthritis develops within a 12-h timeframe, to determine the relative importance of FCGRs and C5 (Hc). In CAIA, inhibiting or deleting FCGR3 substantially hindered arthritis development, underscoring the crucial role of this receptor. Blocking FCGR3 also reduced the levels of FCGR4, and vice versa. When employing an IgG1 arthritogenic cocktail that exclusively interacts with FCGR2B and FCGR3, joint inflammation was promptly initiated in Fcgr2b-- mice but not in Fcgr3-- mice, suggesting that FCGR3 is sufficient for CAIA development. Regarding complement activation, Fcgr2b++.Hc** mice with C5 mutated were fully resistant to CAIA, whereas Fcgr2b--.Hc** mice developed arthritis rapidly. We conclude that FCGR3 is essential and sufficient for CAIA development, particularly when induced by IgG1 antibodies. The human ortholog of mouse FCGR3, FCGR2A, may be associated with RA pathogenesis. FCGR2B deficiency allows for rapid arthritis progression and overrides the resistance conferred by C5 deficiency.
Subject(s)
Arthritis, Experimental , Arthritis, Rheumatoid , Animals , Mice , Cartilage/pathology , Complement C5/genetics , Immunoglobulin G , Receptors, IgG/geneticsABSTRACT
Cranial neural crest cell (NCC)-derived chondrocyte precursors undergo a dynamic differentiation and maturation process to establish a scaffold for subsequent bone formation, alterations in which contribute to congenital birth defects. Here, we demonstrate that transcription factor and histone methyltransferase proteins Prdm3 and Prdm16 control the differentiation switch of cranial NCCs to craniofacial cartilage. Loss of either paralog results in hypoplastic and disorganized chondrocytes due to impaired cellular orientation and polarity. We show that these proteins regulate cartilage differentiation by controlling the timing of Wnt/ß-catenin activity in strikingly different ways: Prdm3 represses whereas Prdm16 activates global gene expression, although both act by regulating Wnt enhanceosome activity and chromatin accessibility. Finally, we show that manipulating Wnt/ß-catenin signaling pharmacologically or generating prdm3-/-;prdm16-/- double mutants rescues craniofacial cartilage defects. Our findings reveal upstream regulatory roles for Prdm3 and Prdm16 in cranial NCCs to control Wnt/ß-catenin transcriptional activity during chondrocyte differentiation to ensure proper development of the craniofacial skeleton.
Subject(s)
Cell Differentiation , MDS1 and EVI1 Complex Locus Protein/metabolism , Wnt Signaling Pathway/genetics , Zebrafish Proteins/metabolism , Animals , Cartilage/cytology , Cartilage/metabolism , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrogenesis , Chromatin/metabolism , Chromatin Assembly and Disassembly , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Developmental , MDS1 and EVI1 Complex Locus Protein/deficiency , MDS1 and EVI1 Complex Locus Protein/genetics , Mice , Mice, Knockout , Neural Crest/cytology , Neural Crest/metabolism , Regulatory Sequences, Nucleic Acid , Skull/cytology , Skull/metabolism , Wnt Proteins/metabolism , Zebrafish , Zebrafish Proteins/deficiency , Zebrafish Proteins/genetics , beta Catenin/metabolismABSTRACT
Osteoarthritis (OA) is a degenerative disease that affects millions of individuals worldwide. Despite its prevalence, the exact causes and mechanisms behind OA are still not fully understood, resulting in a lack of effective treatments to slow down or halt disease progression. Recent research has discovered that extracellular vesicles (EVs) present in the circulation of young mice have a remarkable ability to activate musculoskeletal stem cells in elderly mice. Conversely, EVs derived from elderly mice do not exhibit the same potential, indicating that EVs obtained from young individuals may hold promise to activate aging cells in degenerative tissue. However, it remains unknown whether EVs derived from young individuals can also address cartilage degeneration caused by aging. In this study, we first evaluated EVs derived from young human plasma (YEVs) and EVs derived from old human plasma (OEVs) in an in vitro experiment using chondrocytes. The results revealed that YEVs effectively stimulated chondrocyte proliferation and migration, while OEVs from old plasma did not exhibit a similar effect. Given that OA represents a more complex inflammatory microenvironment, we further determine whether the benefits of YEVs on chondrocytes can be maintained in this context. Our findings indicate that YEVs have the ability to positively regulate chondrocyte function and protect them against apoptosis induced by IL-1ß and TNF-α in an in vitro OA model. Furthermore, we discovered that lyophilized EVs could be stored under mild conditions without any alterations in their physical characteristics. Considering the exceptional therapeutic effects and the wide availability of EVs from young plasma, they hold significant promise as a potential approach to activate chondrocytes and promote cartilage regeneration in early-stage OA.
Subject(s)
Extracellular Vesicles , Osteoarthritis , Humans , Mice , Animals , Chondrocytes , Tumor Necrosis Factor-alpha/pharmacology , Cartilage , Interleukin-1beta/pharmacologyABSTRACT
Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.
Subject(s)
Chondrocytes/cytology , Clone Cells/cytology , Growth Plate/cytology , Stem Cell Niche/physiology , Aging , Animals , Cartilage/cytology , Cell Self Renewal , Clone Cells/metabolism , Female , Growth Plate/metabolism , Male , Mechanistic Target of Rapamycin Complex 1/metabolism , MiceABSTRACT
Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes1. Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment1,2. In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms3-6. It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.
Subject(s)
Bone Diseases/metabolism , Bone Diseases/pathology , Chondrocytes/metabolism , Collagen/biosynthesis , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Animals , Cartilage/metabolism , Extracellular Matrix/metabolism , Glucose/metabolism , Glutamine/metabolism , Growth Plate/metabolism , Hydroxylation , Hypoxia-Inducible Factor-Proline Dioxygenases/deficiency , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Ketoglutaric Acids/metabolism , Lysine/metabolism , Male , Mice , Osteogenesis , Oxidation-Reduction , Proline/metabolismABSTRACT
Chondrogenesis is a multistep process, in which cartilage progenitor cells generate a tissue with distinct structural and functional properties. Although several approaches to cartilage regeneration rely on the differentiation of implanted progenitor cells, the temporal transcriptomic landscape of in vitro chondrogenesis in different models has not been reported. Using RNA sequencing, we examined differences in gene expression patterns during cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. Principal component and trajectory analyses revealed a progressively different and distinct transcriptome during chondrogenesis. Differentially expressed genes (DEGs), based on pairwise comparisons of samples from consecutive days were classified into clusters and analysed. We confirmed the involvement of the top DEGs in chondrogenic differentiation using pathway analysis and identified several chondrogenesis-associated transcription factors and collagen subtypes that were not previously linked to cartilage formation. Transient gene silencing of ATOH8 or EBF1 on day 0 attenuated chondrogenesis by deregulating the expression of key osteochondrogenic marker genes in micromass cultures. These results provide detailed insight into the molecular mechanism of chondrogenesis in primary micromass cultures and present a comprehensive dataset of the temporal transcriptomic landscape of chondrogenesis, which may serve as a platform for new molecular approaches in cartilage tissue engineering.
Subject(s)
Chondrogenesis , Transcriptome , Chondrogenesis/genetics , Cartilage/metabolism , Cell Differentiation/genetics , Stem Cells/metabolism , Cells, Cultured , Chondrocytes/metabolismABSTRACT
The mandible is composed of several musculoskeletal tissues including bone, cartilage, and tendon that require precise patterning to ensure structural and functional integrity. Interestingly, most of these tissues are derived from one multipotent cell population called cranial neural crest cells (CNCCs). How CNCCs are properly instructed to differentiate into various tissue types remains nebulous. To better understand the mechanisms necessary for the patterning of mandibular musculoskeletal tissues we utilized the avian mutant talpid2 (ta2) which presents with several malformations of the facial skeleton including dysplastic tendons, mispatterned musculature, and bilateral ectopic cartilaginous processes extending off Meckel's cartilage. We found an ectopic epithelial BMP signaling domain in the ta2 mandibular prominence (MNP) that correlated with the subsequent expansion of SOX9+ cartilage precursors. These findings were validated with conditional murine models suggesting an evolutionarily conserved mechanism for CNCC-derived musculoskeletal patterning. Collectively, these data support a model in which cilia are required to define epithelial signal centers essential for proper musculoskeletal patterning of CNCC-derived mesenchyme.