Progenitor-like cells contributing to cellular heterogeneity in the nucleus pulposus are lost in intervertebral disc degeneration.

The nucleus pulposus (NP) in the intervertebral disc (IVD) arises from embryonic notochord. Loss of notochordal-like cells in humans correlates with onset of IVD degeneration, suggesting that they are critical for healthy NP homeostasis and function. Comparative transcriptomic analyses identified expression of progenitor-associated genes (GREM1, KRT18, and TAGLN) in the young mouse and non-degenerated human NP, with TAGLN expression reducing with aging. Lineage tracing using Tagln-CreERt2 mice identified peripherally located proliferative NP (PeriNP) cells in developing and postnatal NP that provide a continuous supply of cells to the entire NP. PeriNP cells were diminished in aged mice and absent in puncture-induced degenerated discs. Single-cell transcriptomes of postnatal Tagln-CreERt2 IVD cells indicate enrichment for TGF-ß signaling in Tagln descendant NP sub-populations. Notochord-specific removal of TGF-ß/BMP mediator Smad4 results in loss of Tagln+ cells and abnormal NP morphologies. We propose Tagln+ PeriNP cells are potential progenitors crucial for NP homeostasis.

A single-cell atlas of conventional central chondrosarcoma reveals the role of endoplasmic reticulum stress in malignant transformation.

The transformation of benign lesions to malignant tumours is a crucial aspect of understanding chondrosarcomas, which are malignant cartilage tumours that could develop from benign chondroid lesions. However, the process of malignant transformation for chondroid lesions remains poorly understood, and no reliable markers are available to aid clinical decision-making. To address this issue, we conducted a study analysing 11 primary cartilage tumours and controls using single-cell RNA sequencing. By creating a single-cell atlas, we were able to identify the role of endoplasmic reticulum (ER) stress in the malignant transformation of conventional central chondrosarcomas (CCCS). Our research revealed that lower levels of ER stress promote chondrosarcoma growth in a patient-derived xenograft mouse model, while intensive ER stress reduces primary chondrosarcoma cell viability. Furthermore, we discovered that the NF-κB pathway alleviates ER stress-induced apoptosis during chondrosarcoma progression. Our single-cell signatures and large public data support the use of key ER stress regulators, such as DNA Damage Inducible Transcript 3 (DDIT3; also known as CHOP), as malignant markers for overall patient survival. Ultimately, our study highlights the significant role that ER stress plays in the malignant transformation of cartilaginous tumours and provides a valuable resource for future diagnostic markers and therapeutic strategies.

MMP14 cleaves PTH1R in the chondrocyte-derived osteoblast lineage, curbing signaling intensity for proper bone anabolism.

Bone homeostasis is regulated by hormones such as parathyroid hormone (PTH). While PTH can stimulate osteo-progenitor expansion and bone synthesis, how the PTH-signaling intensity in progenitors is controlled is unclear. Endochondral bone osteoblasts arise from perichondrium-derived osteoprogenitors and hypertrophic chondrocytes (HC). We found, via single-cell transcriptomics, that HC-descendent cells activate membrane-type 1 metalloproteinase 14 (MMP14) and the PTH pathway as they transition to osteoblasts in neonatal and adult mice. Unlike Mmp14 global knockouts, postnatal day 10 (p10) HC lineage-specific Mmp14 null mutants (Mmp14ΔHC) produce more bone. Mechanistically, MMP14 cleaves the extracellular domain of PTH1R, dampening PTH signaling, and consistent with the implied regulatory role, in Mmp14ΔHC mutants, PTH signaling is enhanced. We found that HC-derived osteoblasts contribute ~50% of osteogenesis promoted by treatment with PTH 1-34, and this response was amplified in Mmp14ΔHC. MMP14 control of PTH signaling likely applies also to both HC- and non-HC-derived osteoblasts because their transcriptomes are highly similar. Our study identifies a novel paradigm of MMP14 activity-mediated modulation of PTH signaling in the osteoblast lineage, contributing new insights into bone metabolism with therapeutic significance for bone-wasting diseases.

Hypomorphic and dominant-negative impact of truncated SOX9 dysregulates Hedgehog-Wnt signaling, causing campomelia.

Haploinsufficiency for SOX9, the master chondrogenesis transcription factor, can underlie campomelic dysplasia (CD), an autosomal dominant skeletal malformation syndrome, because heterozygous Sox9 null mice recapitulate the bent limb (campomelia) and some other phenotypes associated with CD. However, in vitro cell assays suggest haploinsufficiency may not apply for certain mutations, notably those that truncate the protein, but in these cases in vivo evidence is lacking and underlying mechanisms are unknown. Here, using conditional mouse mutants, we compared the impact of a heterozygous Sox9 null mutation (Sox9+/-) with the Sox9+/Y440X CD mutation that truncates the C-terminal transactivation domain but spares the DNA-binding domain. While some Sox9+/Y440X mice survived, all Sox9+/- mice died perinatally. However, the skeletal defects were more severe and IHH signaling in developing limb cartilage was significantly enhanced in Sox9+/Y440X compared with Sox9+/-. Activating Sox9Y440X specifically in the chondrocyte-osteoblast lineage caused milder campomelia, and revealed cell- and noncell autonomous mechanisms acting on chondrocyte differentiation and osteogenesis in the perichondrium. Transcriptome analyses of developing Sox9+/Y440X limbs revealed dysregulated expression of genes for the extracellular matrix, as well as changes consistent with aberrant WNT and HH signaling. SOX9Y440X failed to interact with ß-catenin and was unable to suppress transactivation of Ihh in cell-based assays. We propose enhanced HH signaling in the adjacent perichondrium induces asymmetrically localized excessive perichondrial osteogenesis resulting in campomelia. Our study implicates combined haploinsufficiency/hypomorphic and dominant-negative actions of SOX9Y440X, cell-autonomous and noncell autonomous mechanisms, and dysregulated WNT and HH signaling, as the cause of human campomelia.

SOX9 and SOX10 control fluid homeostasis in the inner ear for hearing through independent and cooperative mechanisms.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

Hedgehog signaling orchestrates cartilage-to-bone transition independently of Smoothened.

Although recent lineage studies strongly support a chondrocyte-to-osteoblast differentiation continuum, the biological significance and molecular basis remain undetermined. In silico analysis at a single-cell level indicates a transient shutdown of Hedgehog-related transcriptome during simulated cartilage-to-bone transition. Prompted by this, we genetically induce gain- and loss-of function to probe the role of Hedgehog signaling in cartilage-to-bone transition. Ablating Smo in hypertrophic chondrocytes (HCs) does not result in any phenotypic outcome, whereas deleting Ptch1 in HCs leads to disrupted formation of primary spongiosa and actively proliferating HCs-derived osteogenic cells that contribute to bony bulges seen in adult mutant mice. In HCs-derived osteoblasts, constitutive activation of Hedgehog signaling blocks their further differentiation to osteocytes. Moreover, ablation of both Smo and Ptch1 in HCs reverses neither persistent Hedgehog signaling nor bone overgrowths. These results establish a functional contribution of extended chondrocyte lineage to bone homeostasis and diseases, governed by an unanticipated mode of regulation for Hedgehog signaling independently of Smo.

Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development.

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow-associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; Rosa26fs-tdTomato and Col10a1Cre; Rosa26fs-tdTomato) in combination with a PDGFRaH2B-GFP transgenic line, single-cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow-associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte-derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

4PBA reduces growth deficiency in osteogenesis imperfecta by enhancing transition of hypertrophic chondrocytes to osteoblasts.

Short stature is a major skeletal phenotype in osteogenesis imperfecta (OI), a genetic disorder mainly caused by mutations in genes encoding type I collagen. However, the underlying mechanism is poorly understood, and no effective treatment is available. In OI mice that carry a G610C mutation in COL1A2, we previously found that mature hypertrophic chondrocytes (HCs) are exposed to cell stress due to accumulation of misfolded mutant type I procollagen in the endoplasmic reticulum (ER). By fate mapping analysis of HCs in G610C OI mice, we found that HCs stagnate in the growth plate, inhibiting translocation of HC descendants to the trabecular area and their differentiation to osteoblasts. Treatment with 4-phenylbutyric acid (4PBA), a chemical chaperone, restored HC ER structure and rescued this inhibition, resulting in enhanced longitudinal bone growth in G610C OI mice. Interestingly, the effects of 4PBA on ER dilation were limited in osteoblasts, and the bone fragility was not ameliorated. These results highlight the importance of targeting HCs to treat growth deficiency in OI. Our findings demonstrate that HC dysfunction induced by ER disruption plays a critical role in the pathogenesis of OI growth deficiency, which lays the foundation for developing new therapies for OI.

Integrative analysis of metabolomic, genomic, and imaging-based phenotypes identify very-low-density lipoprotein as a potential risk factor for lumbar Modic changes.

PURPOSE: Modic changes (MC) on magnetic resonance imaging (MRI) have been associated with the development and severity of low back pain (LBP). The etiology of MC remains elusive, but it has been suggested that altered metabolism may be a risk factor. As such, this study aimed to identify metabolomic biomarkers for MC phenotypes of the lumbar spine via a combined metabolomic-genomic approach. METHODS: A population cohort of 3,584 southern Chinese underwent lumbar spine MRI. Blood samples were genotyped with single-nucleotide polymorphisms (SNP) arrays (n = 2,482) and serum metabolomics profiling using magnetic resonance spectroscopy (n = 757), covering 130 metabolites representing three molecular windows, were assessed. Genome-wide association studies (GWAS) were performed on each metabolite, to construct polygenic scores for predicting metabolite levels in subjects who had GWAS but not metabolomic data. Associations between predicted metabolite levels and MC phenotypes were assessed using linear/logistic regression and least absolute shrinkage and selection operator (LASSO). Two-sample Mendelian randomization analysis tested for causal relationships between metabolic biomarkers and MC. RESULTS: 20.4% had MC (10.6% type 1, 67.2% type 2, 22.2% mixed types). Significant MC metabolomic biomarkers were mean diameter of very-low-density lipoprotein (VLDL)/low-density lipoprotein (LDL) particles and cholesterol esters/phospholipids in large LDL. Mendelian randomization indicated that decreased VLDL mean diameter may lead to MC. CONCLUSIONS: This large-scale study is the first to address metabolomics in subject with/without lumbar MC. Causality studies implicate VLDL related to MC, noting a metabolic etiology. Our study substantiates the field of "spino-metabolomics" and illustrates the power of integrating metabolomics-genomics-imaging phenotypes to discover biomarkers for spinal disorders, paving the way for more personalized spine care for patients.

PRIMUS: Comprehensive proteomics of mouse intervertebral discs that inform novel biology and relevance to human disease modelling.

Mice are commonly used to study intervertebral disc (IVD) biology and related diseases such as IVD degeneration. Discs from both the lumbar and tail regions are used. However, little is known about compartmental characteristics in the different regions, nor their relevance to the human setting, where a functional IVD unit depends on a homeostatic proteome. Here, we address these major gaps through comprehensive proteomic profiling and in-depth analyses of 8-week-old healthy murine discs, followed by comparisons with human. Leveraging on a dataset of over 2,700 proteins from 31 proteomic profiles, we identified key molecular and cellular differences between disc compartments and spine levels, but not gender. The nucleus pulposus (NP) and annulus fibrosus (AF) compartments differ the most, both in matrisome and cellularity contents. Differences in the matrisome are consistent with the fibrous nature required for tensile strength in the AF and hydration property in the NP. Novel findings for the NP cells included an enrichment in cell junction proteins for cell-cell communication (Cdh2, Dsp and Gja1) and osmoregulation (Slc12a2 and Wnk1). In NP cells, we detected heterogeneity of vacuolar organelles; where about half have potential lysosomal function (Vamp3, Copb2, Lamp1/2, Lamtor1), some contain lipid droplets and others with undefined contents. The AF is enriched in proteins for the oxidative stress responses (Sod3 and Clu). Interestingly, mitochondrial proteins are elevated in the lumbar than tail IVDs that may reflect differences in metabolic requirement. Relative to the human, cellular and structural information are conserved for the AF. Even though the NP is more divergent between mouse and human, there are similarities at the level of cell biology. Further, common cross-species markers were identified for both NP (KRT8/19, CD109) and AF (COL12A1). Overall, mouse is a relevant model to study IVD biology, and an understanding of the limitation will facilitate research planning and data interpretation, maximizing the translation of research findings to human IVDs.

DIPPER, a spatiotemporal proteomics atlas of human intervertebral discs for exploring ageing and degeneration dynamics.

The spatiotemporal proteome of the intervertebral disc (IVD) underpins its integrity and function. We present DIPPER, a deep and comprehensive IVD proteomic resource comprising 94 genome-wide profiles from 17 individuals. To begin with, protein modules defining key directional trends spanning the lateral and anteroposterior axes were derived from high-resolution spatial proteomes of intact young cadaveric lumbar IVDs. They revealed novel region-specific profiles of regulatory activities and displayed potential paths of deconstruction in the level- and location-matched aged cadaveric discs. Machine learning methods predicted a 'hydration matrisome' that connects extracellular matrix with MRI intensity. Importantly, the static proteome used as point-references can be integrated with dynamic proteome (SILAC/degradome) and transcriptome data from multiple clinical samples, enhancing robustness and clinical relevance. The data, findings, and methodology, available on a web interface (http://www.sbms.hku.hk/dclab/DIPPER/), will be valuable references in the field of IVD biology and proteomic analytics.

The backbone of vertebrate animals consists of a series of bones called vertebrae that are joined together by disc-like structures that allow the back to move and distribute forces to protect it during daily activities. It is common for these intervertebral discs to degenerate with age, resulting in back pain and severely reducing quality of life. The mechanical features of intervertebral discs are the result of their proteins. These include extracellular matrix proteins, which form the external scaffolding that binds cells together in a tissue, and signaling proteins, which allow cells to communicate. However, how the levels of different proteins in each region of the disc vary with time has not been fully examined. To establish how protein composition changes with age, Tam, Chen et al. quantified the protein levels and gene activity (which leads to protein production) of intervertebral discs from young and old deceased individuals. They found that the position of different mixtures of proteins in the intervertebral disc changes with age, and that young people have high levels of extracellular matrix proteins and signaling proteins. Levels of these proteins decreased as people got older, as did the amount of proteins produced. To determine which region of the intervertebral disc different proteins were in, Tam, Chen et al. also performed magnetic resonance imaging (MRI) of the samples to correlate image intensity (which represents water content) with the corresponding protein signature. The data obtained provides a high-quality map of how the location of different proteins changes with age, and is available online under the name DIPPER. This database is an informative resource for research into skeletal biology, and it will likely advance the understanding of intervertebral disc degeneration in humans and animals, potentially leading to the development of new treatment strategies for this condition.

ß1 Integrin regulates convergent extension in mouse notogenesis, ensures notochord integrity and the morphogenesis of vertebrae and intervertebral discs.

The notochord drives longitudinal growth of the body axis by convergent extension, a highly conserved developmental process that depends on non-canonical Wnt/planar cell polarity (PCP) signaling. However, the role of cell-matrix interactions mediated by integrins in the development of the notochord is unclear. We developed transgenic Cre mice, in which the ß1 integrin gene (Itgb1) is ablated at E8.0 in the notochord only or in the notochord and tail bud. These Itgb1 conditional mutants display misaligned, malformed vertebral bodies, hemi-vertebrae and truncated tails. From early somite stages, the notochord was interrupted and displaced in these mutants. Convergent extension of the notochord was impaired with defective cell movement. Treatment of E7.25 wild-type embryos with anti-ß1 integrin blocking antibodies, to target node pit cells, disrupted asymmetric localization of VANGL2. Our study implicates pivotal roles of ß1 integrin for the establishment of PCP and convergent extension of the developing notochord, its structural integrity and positioning, thereby ensuring development of the nucleus pulposus and the proper alignment of vertebral bodies and intervertebral discs. Failure of this control may contribute to human congenital spine malformations.

Transformation of resident notochord-descendent nucleus pulposus cells in mouse injury-induced fibrotic intervertebral discs.

Intervertebral disc degeneration (IDD), a major cause of low back pain, occurs with ageing. The core of the intervertebral disc, the nucleus pulposus (NP), embedded in a proteoglycan-rich and gelatinous matrix, is derived from the embryonic notochord. With IDD, the NP becomes fibrous, containing fewer cells, which are fibroblastic and of unknown origin. Here, we used a lineage tracing strategy to investigate the origin of cells in the NP in injury-induced mouse IDD. We established a Foxa2 notochord-specific enhancer-driven Cre transgenic mouse model (Foxa2mNE-Cre) that acts only in the embryonic to foetal period up to E14.5, to genetically label notochord cells with enhanced green fluorescent protein (EGFP). When this mouse is crossed to one carrying a Cre recombinase reporter, Z/EG, EGFP-labelled NP cells are present even at 2 years of age, consistent with their notochordal origin. We induced tail IDD in Foxa2mNE-Cre; Z/EG mice by annulus puncture and observed the degenerative changes for 12 weeks. Soon after puncture, EGFP-labelled NP cells showed strong Col2a1+ expression unlike uninjured control NP. Later, accompanying fibrotic changes, EGFP-positive NP cells expressed fibroblastic and myofibroblastic markers such as Col1a1, ASMA, FAPA and FSP-1. The number of EGFP+ cells co-expressing the fibroblastic markers increased with time after puncture. Our findings suggest resident NP cells initially upregulate Col2a1+ and later transform into fibroblast-like cells during injury-mediated disc degeneration and remodelling. This important discovery concerning the cellular origin of fibrotic pathology in injury-induced IDD has implications for management in disease and ageing.

Deep-learning-assisted biophysical imaging cytometry at massive throughput delineates cell population heterogeneity.

The association of the intrinsic optical and biophysical properties of cells to homeostasis and pathogenesis has long been acknowledged. Defining these label-free cellular features obviates the need for costly and time-consuming labelling protocols that perturb the living cells. However, wide-ranging applicability of such label-free cell-based assays requires sufficient throughput, statistical power and sensitivity that are unattainable with current technologies. To close this gap, we present a large-scale, integrative imaging flow cytometry platform and strategy that allows hierarchical analysis of intrinsic morphological descriptors of single-cell optical and mass density within a population of millions of cells. The optofluidic cytometry system also enables the synchronous single-cell acquisition of and correlation with fluorescently labeled biochemical markers. Combined with deep neural network and transfer learning, this massive single-cell profiling strategy demonstrates the label-free power to delineate the biophysical signatures of the cancer subtypes, to detect rare populations of cells in the heterogeneous samples (10-5), and to assess the efficacy of targeted therapeutics. This technique could spearhead the development of optofluidic imaging cell-based assays that stratify the underlying physiological and pathological processes based on the information-rich biophysical cellular phenotypes.

IRX3 and IRX5 Inhibit Adipogenic Differentiation of Hypertrophic Chondrocytes and Promote Osteogenesis.

Maintaining the correct proportions of different cell types in the bone marrow is critical for bone function. Hypertrophic chondrocytes (HCs) and osteoblasts are a lineage continuum with a minor contribution to adipocytes, but the regulatory network is unclear. Mutations in transcription factors, IRX3 and IRX5, result in skeletal patterning defects in humans and mice. We found coexpression of Irx3 and Irx5 in late-stage HCs and osteoblasts in cortical and trabecular bone. Irx3 and Irx5 null mutants display severe bone deficiency in newborn and adult stages. Quantitative analyses of bone with different combinations of functional alleles of Irx3 and Irx5 suggest these two factors function in a dosage-dependent manner. In Irx3 and Irx5 nulls, the amount of bone marrow adipocytes was increased. In Irx5 nulls, lineage tracing revealed that removal of Irx3 specifically in HCs exacerbated reduction of HC-derived osteoblasts and increased the frequency of HC-derived marrow adipocytes. ß-catenin loss of function and gain of function specifically in HCs affects the expression of Irx3 and Irx5, suggesting IRX3 and IRX5 function downstream of WNT signaling. Our study shows that IRX3 and IRX5 regulate fate decisions in the transition of HCs to osteoblasts and to marrow adipocytes, implicating their potential roles in human skeletal homeostasis and disorders.

Fbxo9 functions downstream of Sox10 to determine neuron-glial fate choice in the dorsal root ganglia through Neurog2 destabilization.

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.

Directed Differentiation of Notochord-like and Nucleus Pulposus-like Cells Using Human Pluripotent Stem Cells.

Intervertebral disc degeneration might be amenable to stem cell therapy, but the required cells are scarce. Here, we report the development of a protocol for directed in vitro differentiation of human pluripotent stem cells (hPSCs) into notochord-like and nucleus pulposus (NP)-like cells of the disc. The first step combines enhancement of ACTIVIN/NODAL and WNT and inhibition of BMP pathways. By day 5 of differentiation, hPSC-derived cells express notochordal cell characteristic genes. After activating the TGF-ß pathway for an additional 15 days, qPCR, immunostaining, and transcriptome data show that a wide array of NP markers are expressed. Transcriptomically, the in vitro-derived cells become more like in vivo adolescent human NP cells, driven by a set of influential genes enriched with motifs bound by BRACHYURY and FOXA2, consistent with an NP cell-like identity. Transplantation of these NP-like cells attenuates fibrotic changes in a rat disc injury model of disc degeneration.

Cellular Plasticity in Musculoskeletal Development, Regeneration, and Disease.

In this review, we highlight themes from a recent workshop focused on "Plasticity of Cell Fate in Musculoskeletal Tissues" held at the Orthopaedic Research Society's 2019 annual meeting. Experts in the field provided examples of mesenchymal cell plasticity during normal musculoskeletal development, regeneration, and disease. A thorough understanding of the biology underpinning mesenchymal cell plasticity may offer a roadmap for promoting regeneration while attenuating pathologic differentiation. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:708-718, 2020.

KIF5B modulates central spindle organization in late-stage cytokinesis in chondrocytes.

BACKGROUND: The growth plate is a special region of the cartilage that drives longitudinal growth of long bones. Proliferating chondrocytes in the growth plate, arranged in columns, divide perpendicular to the long axis of the growth plate then intercalate to re-align with parental columns. Which molecular partners maintain growth plate columnar structures and chondrocyte cytokinesis has not been fully revealed. It is reported that kinesin family member 3A (KIF3A), a subunit of kinesin-2, plays an important role in maintaining columnar organization in growth plates via controlling primary cilia formation and cell proliferation. RESULT: Here we identify kinesin family member 5B (KIF5B), the heavy chain of kinesin-1, a ubiquitously expressed motor protein for anterograde intracellular transport along the microtubule network, as a key modulator of cytokinesis in chondrocytes via maintenance of central spindle organization. We show that KIF5B is concentrated in the central spindle during cytokinesis in both primary chondrocytes and chondrogenic ATDC5 cells. CONCLUSION: The failure of cytokinesis in KIF5B null chondrocytes leads to incomplete cell rotation, disrupting proliferation and differentiation, and results in a disorganized growth plate.

Lgr5 and Col22a1 Mark Progenitor Cells in the Lineage toward Juvenile Articular Chondrocytes.

The synovial joint forms from a pool of progenitor cells in the future region of the joint, the interzone. Expression of Gdf5 and Wnt9a has been used to mark the earliest cellular processes in the formation of the interzone and the progenitor cells. However, lineage specification and progression toward the different tissues of the joint are not well understood. Here, by lineage-tracing studies we identify a population of Lgr5+ interzone cells that contribute to the formation of cruciate ligaments, synovial membrane, and articular chondrocytes of the joint. This finding is supported by single-cell transcriptome analyses. We show that Col22a1, a marker of early articular chondrocytes, is co-expressed with Lgr5+ cells prior to cavitation as an important lineage marker specifying the progression toward articular chondrocytes. Lgr5+ cells contribute to the repair of a joint defect with the re-establishment of a Col22a1-expressing superficial layer.