DNA hypomethylation ameliorates erosive inflammatory arthritis by modulating interferon regulatory factor-8.

Epigenetic regulation plays a crucial role in the pathogenesis of autoimmune diseases such as inflammatory arthritis. DNA hypomethylating agents, such as decitabine (DAC), have been shown to dampen inflammation and restore immune homeostasis. In the present study, we demonstrate that DAC elicits potent anti-inflammatory effects and attenuates disease symptoms in several animal models of arthritis. Transcriptomic and epigenomic profiling show that DAC-mediated hypomethylation regulates a wide range of cell types in arthritis, altering the differentiation trajectories of anti-inflammatory macrophage populations, regulatory T cells, and tissue-protective synovial fibroblasts (SFs). Mechanistically, DAC-mediated demethylation of intragenic 5'-Cytosine phosphate Guanine-3' (CpG) islands of the transcription factor Irf8 (interferon regulatory factor 8) induced its re-expression and promoted its repressor activity. As a result, DAC restored joint homeostasis by resetting the transcriptomic signature of negative regulators of inflammation in synovial macrophages (MerTK, Trem2, and Cx3cr1), TREGs (Foxp3), and SFs (Pdpn and Fapα). In conclusion, we found that Irf8 is necessary for the inhibitory effect of DAC in murine arthritis and that direct expression of Irf8 is sufficient to significantly mitigate arthritis.

Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

Aging and obesity prime the methylome and transcriptome of adipose stem cells for disease and dysfunction.

The epigenome of stem cells occupies a critical interface between genes and environment, serving to regulate expression through modification by intrinsic and extrinsic factors. We hypothesized that aging and obesity, which represent major risk factors for a variety of diseases, synergistically modify the epigenome of adult adipose stem cells (ASCs). Using integrated RNA- and targeted bisulfite-sequencing in murine ASCs from lean and obese mice at 5- and 12-months of age, we identified global DNA hypomethylation with either aging or obesity, and a synergistic effect of aging combined with obesity. The transcriptome of ASCs in lean mice was relatively stable to the effects of age, but this was not true in obese mice. Functional pathway analyses identified a subset of genes with critical roles in progenitors and in diseases of obesity and aging. Specifically, Mapt, Nr3c2, App, and Ctnnb1 emerged as potential hypomethylated upstream regulators in both aging and obesity (AL vs. YL and AO vs. YO), and App, Ctnnb1, Hipk2, Id2, and Tp53 exhibited additional effects of aging in obese animals. Furthermore, Foxo3 and Ccnd1 were potential hypermethylated upstream regulators of healthy aging (AL vs. YL), and of the effects of obesity in young animals (YO vs. YL), suggesting that these factors could play a role in accelerated aging with obesity. Finally, we identified candidate driver genes that appeared recurrently in all analyses and comparisons undertaken. Further mechanistic studies are needed to validate the roles of these genes capable of priming ASCs for dysfunction in aging- and obesity-associated pathologies.

Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

Chondrogenic Differentiation of Human-Induced Pluripotent Stem Cells.

The generation of large quantities of genetically defined human chondrocytes remains a critical step for the development of tissue engineering strategies for cartilage regeneration and high-throughput drug screening. This protocol describes chondrogenic differentiation of human-induced pluripotent stem cells (hiPSCs), which can undergo genetic modification and the capacity for extensive cell expansion. The hiPSCs are differentiated in a stepwise manner in monolayer through the mesodermal lineage for 12 days using defined growth factors and small molecules. This is followed by 28 days of chondrogenic differentiation in a 3D pellet culture system using transforming growth factor beta 3 and specific compounds to inhibit off-target differentiation. The 6-week protocol results in hiPSC-derived cartilaginous tissue that can be characterized by histology, immunohistochemistry, and gene expression or enzymatically digested to isolate chondrocyte-like cells. Investigators can use this protocol for experiments including genetic engineering, in vitro disease modeling, or tissue engineering.

Whole-genome RNA sequencing identifies distinct transcriptomic profiles in impingement cartilage between patients with femoroacetabular impingement and hip osteoarthritis.

Femoroacetabular impingement (FAI) has a strong clinical association with the development of hip osteoarthritis (OA); however, the pathobiological mechanisms underlying the transition from focal impingement to global joint degeneration remain poorly understood. The purpose of this study is to use whole-genome RNA sequencing to identify and subsequently validate differentially expressed genes (DEGs) in femoral head articular cartilage samples from patients with FAI and hip OA secondary to FAI. Thirty-seven patients were included in the study with whole-genome RNA sequencing performed on 10 gender-matched patients in the FAI and OA cohorts and the remaining specimens were used for validation analyses. We identified a total of 3531 DEGs between the FAI and OA cohorts with multiple targets for genes implicated in canonical OA pathways. Quantitative reverse transcription-polymerase chain reaction validation confirmed increased expression of FGF18 and WNT16 in the FAI samples, while there was increased expression of MMP13 and ADAMTS4 in the OA samples. Expression levels of FGF18 and WNT16 were also higher in FAI samples with mild cartilage damage compared to FAI samples with severe cartilage damage or OA cartilage. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. We independently validated the results of the sequencing analysis and found increased expression of anabolic markers in patients with FAI and minimal histologic cartilage damage, suggesting that anabolic signaling may be increased in early FAI with a transition to catabolic and inflammatory gene expression as FAI progresses towards more severe hip OA. Clinical significance:Cam-type FAI has a strong clinical association with hip OA; however, the cellular pathophysiology of disease progression remains poorly understood. Several previous studies have demonstrated increased expression of inflammatory markers in FAI cartilage samples, suggesting the involvement of these inflammatory pathways in the disease progression. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. In addition to differences in inflammatory gene expression, we also identified differential expression in multiple pathways involved in hip OA progression.

Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 in osteoarthritis.

OBJECTIVES: Synovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/ß-catenin signalling, given its emerging importance in arthritis. METHODS: We subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain of function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. RESULTS: We uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/ß-catenin signalling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages and chondrocytes. CONCLUSIONS: Synovial fibroblasts assume distinct functional identities during PTOA in mice, and Prg4hi lining fibroblasts secrete Rspo2 that may drive pathological joint crosstalk after injury.

The surgical destabilization of the abductor muscle leads to development of instability-associated hip osteoarthritis in mice.

Osteoarthritis (OA) of the hip is a common and debilitating painful joint disease. However, there is paucity of surgically induced hip OA models in small animals that allow scientists to study the onset and progression of the disease. A growing body of evidence indicates a positive association between periarticular myotendinous pathology and the development of hip OA. Thus, in the current study, we aimed to establish a novel mouse instability-associated hip OA model via selective injury of the abductor complex around the hip joint. C57BL6/J mice were randomized to sham surgery or abductor injury, in which the myotendinous insertion at the third trochanter and greater trochanter were surgically detached. Mice were allowed free active movement until they were sacrificed at either 3 weeks or 20 weeks post-injury. Histologic analyses and immunohistochemical staining of the femoral head articular cartilage were performed, along with microCT (µCT) analysis to assess subchondral bone remodeling. We observed that mice receiving abductor injury exhibited significantly increased instability-associated OA severity with loss of proteoglycan and type II collagen staining compared to sham control mice at 20 weeks post-surgery, while comparable matrix metalloproteinase 13 expression was observed between injury and sham groups. No significant differences in subchondral bone remodeling were found after 3 or 20 weeks following injury. Our study further supports the link between abductor dysfunction and the development of instability-associated hip OA. Importantly, this novel surgically induced hip OA mouse model may provide a valuable tool for future investigations into the pathogenesis and treatment of hip OA.

Defining the spatial-molecular map of fibrotic tendon healing and the drivers of Scleraxis-lineage cell fate and function.

Tendon injuries heal via a scar-mediated response, and there are no biological approaches to promote more regenerative healing. Mouse flexor tendons heal through the formation of spatially distinct tissue areas: a highly aligned tissue bridge between the native tendon stubs that is enriched for adult Scleraxis-lineage cells and a disorganized outer shell associated with peri-tendinous scar formation. However, the specific molecular programs that underpin these spatially distinct tissue profiles are poorly defined. In the present study, we combine lineage tracing of adult Scleraxis-lineage cells with spatial transcriptomic profiling to define the overarching molecular programs that govern tendon healing and cell-fate decisions. Pseudotime analysis identified three fibroblast trajectories (synthetic, fibrotic, and reactive) and key transcription factors regulating these fate-switching decisions, including the progression of adult Scleraxis-lineage cells through the reactive trajectory. Collectively, this resource defines the molecular mechanisms that coordinate the temporo-spatial healing phenotype, which can be leveraged to inform therapeutic candidate selection.

Single-cell transcriptomics of popliteal lymphatic vessels and peripheral veins reveals altered lymphatic muscle and immune cell populations in the TNF-Tg arthritis model.

BACKGROUND: Lymphatic dysfunction exists in tumor necrosis factor transgenic (TNF-Tg) mice and rheumatoid arthritis (RA) patients. While joint-draining TNF-Tg popliteal lymphatic vessels (PLVs) have deficits in contractility during end-stage arthritis, the nature of lymphatic muscle cells (LMCs) and their TNF-altered transcriptome remain unknown. Thus, we performed single-cell RNA-sequencing (scRNAseq) on TNF-Tg LMCs in PLVs efferent to inflamed joints versus wild-type (WT) controls. METHODS: Single-cell suspensions of PLVs were sorted for smooth muscle cells (SMCs), which was validated by Cspg4-Cre;tdTomato reporter gene expression. Single-cell RNA-seq was performed on a 10x Genomics platform and analyzed using the Seurat R package. Uniform Manifold Approximation and Projections (UMAPs) and Ingenuity Pathway Analysis software were used to assess cell clusters and functional genomics in WT vs. TNF-Tg populations. RESULTS: Fluorescent imaging of Cspg4-Cre;tdTomato vessels demonstrated dim PLVs and strong reporter gene expression in the adjacent superficial saphenous vein, which was corroborated by flow cytometry of LMCs and vascular smooth muscle cells (VSMCs) from these vessels. Due to their unique morphology, these populations could also be readily detected by scatter analysis of cells from non-fluorescent mice. Bioinformatics analysis of flow sorted WT and TNF-Tg cells identified 20 unique cell clusters that together were 22.4% LMCs, 15.0% VSMCs, and 62.6% non-muscle cells of 8879 total cells. LMCs and M2-macrophages were decreased, while inflammatory monocytes were increased in TNF-Tg lower limb vasculature. SMC populations were defined by Cald1, Tpm1, and Pdgfrb expression and were enriched in myofibroblast-like gene expression. TNF-Tg LMCs exhibited enhanced functional genomics associated with cell death, phagocyte recruitment, and joint inflammation. Among the most prominent TNF-induced genes in SMCs were Mmp3, Cxcl12, and Ccl19, and the most downregulated genes were Zbtb16, Galnt15, and Apod. CONCLUSIONS: Single-cell RNA-seq can be used to investigate functional genomics of lower limb vasculature in mice. Our findings confirm the inflammatory transcriptome of TNF-Tg vessels and altered gene expression in SMC populations. This study further supports a potential role of mesenchymal stromal cells in inflammatory-erosive arthritis pathogenesis, and warrants future studies to define the effects of this TNF-altered transcriptome on PLV function and joint homeostasis.

A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery.

Biologic drug therapies are increasingly used for inflammatory diseases such as rheumatoid arthritis but may cause significant adverse effects when delivered continuously at high doses. We used CRISPR-Cas9 genome editing of iPSCs to create a synthetic gene circuit that senses changing levels of endogenous inflammatory cytokines to trigger a proportional therapeutic response. Cells were engineered into cartilaginous constructs that showed rapid activation and recovery in response to inflammation in vitro or in vivo. In the murine K/BxN model of inflammatory arthritis, bioengineered implants significantly mitigated disease severity as measured by joint pain, structural damage, and systemic and local inflammation. Therapeutic implants completely prevented increased pain sensitivity and bone erosions, a feat not achievable by current clinically available disease-modifying drugs. Combination tissue engineering and synthetic biology promises a range of potential applications for treating chronic diseases via custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.

Transient receptor potential vanilloid 4 as a regulator of induced pluripotent stem cell chondrogenesis.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal calcium-permeable cation channel that is highly expressed in cartilage and is sensitive to a variety of extracellular stimuli. The expression of this channel has been associated with the process of chondrogenesis in adult stem cells as well as several cell lines. Here, we used a chondrogenic reporter (Col2a1-GFP) in murine induced pluripotent stem cells (iPSCs) to examine the hypothesis that TRPV4 serves as both a marker and a regulator of chondrogenesis. Over 21 days of chondrogenesis, iPSCs showed significant increases in Trpv4 expression along with the standard chondrogenic gene markers Sox9, Acan, and Col2a1, particularly in the green fluorescent protein positive (GFP+) chondroprogenitor subpopulation. Increased gene expression for Trpv4 was also reflected by the presence of TRPV4 protein and functional Ca2+ signaling. Daily activation of TRPV4 using the specific agonist GSK1016790A resulted in significant increases in cartilaginous matrix production. An improved understanding of the role of TRPV4 in chondrogenesis may provide new insights into the development of new therapeutic approaches for diseases of cartilage, such as osteoarthritis, or channelopathies and hereditary disorders that affect cartilage during development. Harnessing the role of TRPV4 in chondrogenesis may also provide a novel approach for accelerating stem cell differentiation in functional tissue engineering of cartilage replacements for joint repair.

Single Cell Omics for Musculoskeletal Research.

PURPOSE OF REVIEW: The ability to analyze the molecular events occurring within individual cells as opposed to populations of cells is revolutionizing our understanding of musculoskeletal tissue development and disease. Single cell studies have the great potential of identifying cellular subpopulations that work in a synchronized fashion to regenerate and repair damaged tissues during normal homeostasis. In addition, such studies can elucidate how these processes break down in disease as well as identify cellular subpopulations that drive the disease. This review highlights three emerging technologies: single cell RNA sequencing (scRNA-seq), Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), and Cytometry by Time-Of-Flight (CyTOF) mass cytometry. RECENT FINDINGS: Technological and bioinformatic tools to analyze the transcriptome, epigenome, and proteome at the individual cell level have advanced rapidly making data collection relatively easy; however, understanding how to access and interpret the data remains a challenge for many scientists. It is, therefore, of paramount significance to educate the musculoskeletal community on how single cell technologies can be used to answer research questions and advance translation. This article summarizes talks given during a workshop on "Single Cell Omics" at the 2020 annual meeting of the Orthopedic Research Society. Studies that applied scRNA-seq, ATAC-seq, and CyTOF mass cytometry to cartilage development and osteoarthritis are reviewed. This body of work shows how these cutting-edge tools can advance our understanding of the cellular heterogeneity and trajectories of lineage specification during development and disease.

Single-cell RNA sequencing reveals the induction of novel myeloid and myeloid-associated cell populations in visceral fat with long-term obesity.

Macrophages and other immune cells are important contributors to obesity-associated inflammation; however, the cellular identities of these specific populations remain unknown. In this study, we identified individual populations of myeloid cells found in mouse epididymal/visceral adipose tissue by single-cell RNA sequencing, immunofluorescence, and flow cytometry. Multiple canonical correlation analysis identified 11 unique myeloid and myeloid-associate cell populations. In obese mice, we detected an increased percentage of monocyte-derived pro-inflammatory cells expressing Cd9 and Trem2, as well as significantly decreased percentages of multiple cell populations, including tissue-resident cells expressing Lyve1, Mafb, and Mrc1. We have identified and validated a novel myeloid/macrophage population defined by Ly6a expression, exhibiting both myeloid and mesenchymal characteristics, which increased with obesity and showed high pro-fibrotic characteristics in vitro. Our mouse adipose tissue myeloid cell atlas provides an important resource to investigate obesity-associated inflammation and fibrosis.

Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis.

The therapeutic application of human induced pluripotent stem cells (hiPSCs) for cartilage regeneration is largely hindered by the low yield of chondrocytes accompanied by unpredictable and heterogeneous off-target differentiation of cells during chondrogenesis. Here, we combine bulk RNA sequencing, single cell RNA sequencing, and bioinformatic analyses, including weighted gene co-expression analysis (WGCNA), to investigate the gene regulatory networks regulating hiPSC differentiation under chondrogenic conditions. We identify specific WNTs and MITF as hub genes governing the generation of off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. With heterocellular signaling models, we further show that WNT signaling produced by off-target cells is responsible for inducing chondrocyte hypertrophy. By targeting WNTs and MITF, we eliminate these cell lineages, significantly enhancing the yield and homogeneity of hiPSC-derived chondrocytes. Collectively, our findings identify the trajectories and molecular mechanisms governing cell fate decision in hiPSC chondrogenesis, as well as dynamic transcriptome profiles orchestrating chondrocyte proliferation and differentiation.

Is Obesity a Disease of Stem Cells?

Obesity disrupts physiological homeostasis and alters both systemic and local microenvironments that impact stem cell plasticity and impair regenerative capacity. We present growing evidence that reveals the bidirectionality of obesity-induced stem cell dysfunction and how the molecular changes in stem cells residing in obese environments may accelerate disease severity.

Gene therapy for follistatin mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat diet-induced obesity.

Obesity-associated inflammation and loss of muscle function play critical roles in the development of osteoarthritis (OA); thus, therapies that target muscle tissue may provide novel approaches to restoring metabolic and biomechanical dysfunction associated with obesity. Follistatin (FST), a protein that binds myostatin and activin, may have the potential to enhance muscle formation while inhibiting inflammation. Here, we hypothesized that adeno-associated virus 9 (AAV9) delivery of FST enhances muscle formation and mitigates metabolic inflammation and knee OA caused by a high-fat diet in mice. AAV-mediated FST delivery exhibited decreased obesity-induced inflammatory adipokines and cytokines systemically and in the joint synovial fluid. Regardless of diet, mice receiving FST gene therapy were protected from post-traumatic OA and bone remodeling induced by joint injury. Together, these findings suggest that FST gene therapy may provide a multifactorial therapeutic approach for injury-induced OA and metabolic inflammation in obesity.

Transgenic conversion of ω-6 to ω-3 polyunsaturated fatty acids via fat-1 reduces the severity of post-traumatic osteoarthritis.

BACKGROUND: Dietary fatty acid (FA) content has been shown to influence the development of post-traumatic osteoarthritis (PTOA) in obesity. We used the fat-1 transgenic mouse to examine the hypothesis that endogenous reduction of ω-6 to ω-3 FA ratio, under the same dietary conditions, would mitigate metabolic inflammation and the pathogenesis of PTOA in obese male and female mice. METHODS: Male and female fat-1 and wild-type littermates were fed either a control diet or an ω-6 FA-rich high-fat diet and underwent destabilization of the medial meniscus (DMM) surgery to induce PTOA. OA severity, synovitis, and osteophyte formation were determined histologically, while biomarker and lipidomic analyses were performed to evaluate levels of adipokines, insulin, pro-/anti-inflammatory cytokines, and FAs in serum and joint synovial fluid. Multivariable models were performed to elucidate the associations of dietary, metabolic, and mechanical factors with PTOA. RESULTS: We found that elevated serum levels of ω-3 FAs in fat-1 mice as compared to wild-type controls fed the same diet resulted in reduced OA and synovitis in a sex- and diet-dependent manner, despite comparable body weights. The fat-1 mice showed trends toward decreased serum pro-inflammatory cytokines and increased anti-inflammatory cytokines. Multivariable analysis for variables predicting OA severity in mice resulted in correlations with serum FA levels, but not with body weight. CONCLUSIONS: This study provides further evidence that circulating FA composition and systemic metabolic inflammation, rather than body weight, may be the major risk factor for obesity-associated OA. We also demonstrate the potential genetic use of ω-3 FA desaturase in mitigating PTOA in obese patients following injury.

Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter.

BACKGROUND: Articular cartilage shows little or no capacity for intrinsic repair, generating a critical need of regenerative therapies for joint injuries and diseases such as osteoarthritis. Human-induced pluripotent stem cells (hiPSCs) offer a promising cell source for cartilage tissue engineering and in vitro human disease modeling; however, off-target differentiation remains a challenge during hiPSC chondrogenesis. Therefore, the objective of this study was to identify cell surface markers that define the true chondroprogenitor population and use these markers to purify iPSCs as a means of improving the homogeneity and efficiency of hiPSC chondrogenic differentiation. METHODS: We used a CRISPR-Cas9-edited COL2A1-GFP knock-in reporter hiPSC line, coupled with a surface marker screen, to identify a novel chondroprogenitor population. Single-cell RNA sequencing was then used to analyze the distinct clusters within the population. An unpaired t test with Welch's correction or an unpaired Kolmogorov-Smirnov test was performed with significance reported at a 95% confidence interval. RESULTS: Chondroprogenitors expressing CD146, CD166, and PDGFRß, but not CD45, made up an average of 16.8% of the total population. Under chondrogenic culture conditions, these triple-positive chondroprogenitor cells demonstrated decreased heterogeneity as measured by single-cell RNA sequencing with fewer clusters (9 clusters in unsorted vs. 6 in sorted populations) closer together. Additionally, there was more robust and homogenous matrix production (unsorted: 1.5 ng/ng vs. sorted: 19.9 ng/ng sGAG/DNA; p < 0.001) with significantly higher chondrogenic gene expression (i.e., SOX9, COL2A1, ACAN; p < 0.05). CONCLUSIONS: Overall, this study has identified a unique hiPSC-derived subpopulation of chondroprogenitors that are CD146+/CD166+/PDGFRß+/CD45- and exhibit high chondrogenic potential, providing a purified cell source for cartilage tissue engineering or disease modeling studies.

Intergenerational Transmission of Diet-Induced Obesity, Metabolic Imbalance, and Osteoarthritis in Mice.

OBJECTIVE: Obesity and osteoarthritis (OA) are 2 major public health issues affecting millions of people worldwide. Whereas parental obesity affects the predisposition to diseases such as cancer or diabetes in children, transgenerational influences on musculoskeletal conditions such as OA are poorly understood. This study was undertaken to assess the intergenerational effects of a parental/grandparental high-fat diet on the metabolic and skeletal phenotype, systemic inflammation, and predisposition to OA in 2 generations of offspring in mice. METHODS: Metabolic phenotype and predisposition to OA were investigated in the first and second (F1 and F2) generations of offspring (n = 10-16 mice per sex per diet) bred from mice fed a high-fat diet (HFD) or a low-fat control diet. OA was induced by destabilizing the medial meniscus. OA, synovitis, and adipose tissue inflammation were determined histologically, while bone changes were measured using micro-computed tomography. Serum and synovial cytokines were measured by multiplex assay. RESULTS: Parental high-fat feeding showed an intergenerational effect, with inheritance of increased weight gain (up to 19% in the F1 generation and 9% in F2), metabolic imbalance, and injury-induced OA in at least 2 generations of mice, despite the fact that the offspring were fed the low-fat diet. Strikingly, both F1 and F2 female mice showed an increased predisposition to injury-induced OA (48% higher predisposition in F1 and 19% in F2 female mice fed the HFD) and developed bone microarchitectural changes that were attributable to parental and grandparental high-fat feeding. CONCLUSION: The results of this study reveal a detrimental effect of parental HFD and obesity on the musculoskeletal integrity of 2 generations of offspring, indicating the importance of further investigation of these effects. An improved understanding of the mechanisms involved in the transmissibility of diet-induced changes through multiple generations may help in the development of future therapies that would target the effects of obesity on OA and related conditions.