Nidhi Bhutani

All Publications

• Profiling joint tissues at single-cell resolution: advances and insights. Nature reviews. Rheumatology Pandey, A., Bhutani, N. 2023

  Abstract

  Advances in the profiling of human joint tissues at single-cell resolution have provided unique insights into the organization and function of these tissues in health and disease. Data generated by various single-cell technologies, including single-cell RNA sequencing and cytometry by time-of-flight, have identified the distinct subpopulations that constitute these tissues. These timely studies have provided the building blocks for the construction of single-cell atlases of joint tissues including cartilage, bone and synovium, leading to the identification of developmental trajectories, deciphering of crosstalk between cells and discovery of rare populations such as stem and progenitor cells. In addition, these studies have revealed unique pathogenetic populations that are potential therapeutic targets. The use of these approaches in synovial tissues has helped to identify how distinct cell subpopulations can orchestrate disease initiation and progression and be responsible for distinct pathological outcomes. Additionally, repair of tissues such as cartilage and meniscus remains an unmet medical need, and single-cell methodologies can be invaluable in providing a blueprint for both effective tissue-engineering strategies and therapeutic interventions for chronic joint diseases such as osteoarthritis and rheumatoid arthritis.

  View details for DOI 10.1038/s41584-023-01052-x

  View details for PubMedID 38057475

• TET1 regulates skeletal stem cell (SSC) mediated cartilage regeneration. Arthritis & rheumatology (Hoboken, N.J.) Pandey, A., Hoover, M., Singla, M., Bedi, Y., Storaci, H., Goodman, S. B., Chan, C., Bhutani, N. 2023

  Abstract

  Adult skeletal stem cells (SSC) give rise to chondrocytes, osteocytes and stromal cells as progeny have been shown to contribute to cartilage regeneration in Osteoarthritis (OA). Understanding extrinsic and intrinsic regulators of SSC fate and function can therefore identify putative candidate factors to enhance cartilage regeneration. This study explores how the DNA hydroxymethylase, TET1 regulates SSC function in OA.We investigated the differences in SSC lineage tree and differentiation potential in neonatal and adult Tet1 +/+ and Tet1-/- mice, with and without injury and upon OA induction and progression. Using RNA-seq, the transcriptomic differences between SSC and Bone, cartilage and stromal progenitor cells (BCSP) were identified in Tet1 +/+ mice and Tet1-/- mice.Loss of Tet1 skewed the SSC lineage tree by expanding the SSC pool and enhanced the chondrogenic potential of SSC and BCSP. Tet1 inhibition led to enhanced chondrogenesis in in human SSC and chondroprogenitors (CP) isolated from human cartilage. Importantly, TET1 inhibition in vivo in late stages of a mouse model of Osteoarthritis (OA) led to increased cartilage regeneration. Transcriptomic analyses of SSC and BCSP lacking Tet1 revealed pathway alterations in TGFβ signaling, melatonin degradation and cartilage development associated genes. Lastly, we report that use of hormone melatonin can dampen inflammation and improve cartilage health.While Tet1 is a broad epigenetic regulator, Melatonin can mimic the ability of TET1 inhibition to enhance the chondrogenic ability of skeletal stem cells. Melatonin administration has the potential to be an attractive stem cell based therapy for cartilage regeneration.

  View details for DOI 10.1002/art.42678

  View details for PubMedID 37610277

• A dysfunctional TRPV4-GSK3beta pathway prevents osteoarthritic chondrocytes from sensing changes in extracellular matrix viscoelasticity. Nature biomedical engineering Agarwal, P., Lee, H., Smeriglio, P., Grandi, F., Goodman, S., Chaudhuri, O., Bhutani, N. 2021

  Abstract

  Changes in the composition and viscoelasticity of the extracellular matrix in load-bearing cartilage influence the proliferation and phenotypes of chondrocytes, and are associated with osteoarthritis. However, the underlying molecular mechanism is unknown. Here we show that the viscoelasticity of alginate hydrogels regulates cellular volume in healthy human chondrocytes (with faster stress relaxation allowing cell expansion and slower stress relaxation restricting it) but not in osteoarthritic chondrocytes. Cellular volume regulation in healthy chondrocytes was associated with changes in anabolic gene expression, in the secretion of multiple pro-inflammatory cytokines, and in the modulation of intracellular calcium regulated by the ion-channel protein transient receptor potential cation channel subfamily V member 4 (TRPV4), which controls the phosphorylation of glycogen synthase kinase 3beta (GSK3beta), an enzyme with pleiotropic effects in osteoarthritis. A dysfunctional TRPV4-GSK3beta pathway in osteoarthritic chondrocytes rendered the cells unable to respond to environmental changes in viscoelasticity. Our findings suggest strategies for restoring chondrocyte homeostasis in osteoarthritis.

  View details for DOI 10.1038/s41551-021-00691-3

  View details for PubMedID 33707778

• Inhibition of TET1 prevents the development of osteoarthritis and reveals the 5hmC landscape that orchestrates pathogenesis. Science translational medicine Smeriglio, P. n., Grandi, F. C., Davala, S. n., Masarapu, V. n., Indelli, P. F., Goodman, S. B., Bhutani, N. n. 2020; 12 (539)

  Abstract

  Osteoarthritis (OA) is a degenerative disease of the joint, which results in pain, loss of mobility, and, eventually, joint replacement. Currently, no disease-modifying drugs exist, partly because of the multiple levels at which cartilage homeostasis is disrupted. Recent studies have highlighted the importance of epigenetic dysregulation in OA, sparking interest in the epigenetic modulation for this disease. In our previous work, we characterized a fivefold increase in cytosine hydroxymethylation (5hmC), an oxidized derivative of cytosine methylation (5mC) associated with gene activation, accumulating at OA-associated genes. To test the role of 5hmC in OA, here, we used a mouse model of surgically induced OA and found that OA onset was accompanied by a gain of ~40,000 differentially hydroxymethylated sites before the notable histological appearance of disease. We demonstrated that ten-eleven-translocation enzyme 1 (TET1) mediates the 5hmC deposition because 98% of sites enriched for 5hmC in OA were lost in Tet1-/- mice. Loss of TET1-mediated 5hmC protected the Tet1-/- mice from OA development, including degeneration of the cartilage surface and osteophyte formation, by directly preventing the activation of multiple OA pathways. Loss of TET1 in human OA chondrocytes reduced the expression of the matrix metalloproteinases MMP3 and MMP13 and multiple inflammatory cytokines. Intra-articular injections of a dioxygenases inhibitor, 2-hydroxyglutarate, on mice after surgical induction of OA stalled disease progression. Treatment of human OA chondrocytes with the same inhibitor also phenocopied TET1 loss. Collectively, these data demonstrate that TET1-mediated 5hmC deposition regulates multiple OA pathways and can be modulated for therapeutic intervention.

  View details for DOI 10.1126/scitranslmed.aax2332

  View details for PubMedID 32295898

• Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model. Acta neuropathologica communications Sinha, S., Huang, M. S., Mikos, G., Bedi, Y., Soto, L., Lensch, S., Ayushman, M., Bintu, L., Bhutani, N., Heilshorn, S. C., Yang, F. 2024; 12 (1): 71

  Abstract

  Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However, it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here, we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration, we developed a DIPG-neural assembloid model, which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model, we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover, laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally, these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.

  View details for DOI 10.1186/s40478-024-01765-4

  View details for PubMedID 38706008

  View details for PubMedCentralID 4161623

• Lumen expansion is initially driven by apical actin polymerization followed by osmotic pressure in a human epiblast model. Cell stem cell Indana, D., Zakharov, A., Lim, Y., Dunn, A. R., Bhutani, N., Shenoy, V. B., Chaudhuri, O. 2024; 31 (5): 640-656.e8

  Abstract

  Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. Osmotic pressure gradients are considered the drivers of lumen expansion across development, but their role in human epiblasts is unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based epiblast model using engineered hydrogels. We find that leaky junctions prevent osmotic pressure gradients in early epiblasts and, instead, forces from apical actin polymerization drive lumen expansion. Once the lumen reaches a radius of ∼12 μm, tight junctions mature, and osmotic pressure gradients develop to drive further growth. Computational modeling indicates that apical actin polymerization into a stiff network mediates initial lumen expansion and predicts a transition to pressure-driven growth in larger epiblasts to avoid buckling. Human epiblasts show transcriptional signatures consistent with these mechanisms. Thus, actin polymerization drives lumen expansion in the human epiblast and may serve as a general mechanism of early lumenogenesis.

  View details for DOI 10.1016/j.stem.2024.03.016

  View details for PubMedID 38701758

• Targeting an inflammation-amplifying cell population can attenuate osteoarthritis-associated pain. Arthritis research & therapy Pandey, A., Singla, M., Geller, A., Goodman, S. B., Bhutani, N. 2024; 26 (1): 53

  Abstract

  BACKGROUND: Understanding of pain in osteoarthritis, its genesis, and perception is still in its early stages. Identification of precise ligand-receptor pairs that transduce pain and the cells and tissues in which they reside will elucidate new therapeutic approaches for pain management. Our recent studies had identified an inflammation-amplifying (Inf-A) cell population that is expanded in human OA cartilage and is distinctive in the expression of both IL1R1 and TNF-R2 receptors and active Jnk signaling cascade.METHODS: In this study, we have tested the function of the cartilage-resident IL1R1+TNF-R2+ Inf-A cells in OA. We have identified that the IL1R1+TNF-R2+ Inf-A cells expand in aged mice as well as after anterior cruciate ligament tear upon tibia loading and OA initiation in mice. We targeted and modulated the Jnk signaling cascade in InfA through competitive inhibition of Jnk signaling in mice and human OA explants and tested the effects on joint structure and gait in mice.RESULTS: Modulation of Jnk signaling led to attenuation of inflammatory cytokines CCL2 and CCL7 without showing any structural improvements in the joint architecture. Interestingly, Jnk inhibition and lowered CCL2 and 7 are sufficient to significantly improve the gait parameters in treated PTOA mice demonstrating reduced OA-associated pain. Consistent with the mice data, treatment with JNK inhibitor did not improve human OA cartilage explants.CONCLUSION: These studies demonstrate that Inf-A, an articular-cartilage resident cell population, contributes to pain in OA via secretion of CCL2 and 7 and can be targeted via inhibition of Jnk signaling.

  View details for DOI 10.1186/s13075-024-03284-y

  View details for PubMedID 38368390

• A single-cell mass cytometry platform to map the effects of preclinical drugs on cartilage homeostasis. JCI insight Sahu, N., Grandi, F. C., Bhutani, N. 2022

  Abstract

  No disease-modifying drug exists for osteoarthritis (OA). Despite success in animal models, candidate drugs continue to fail in clinical trials due to the unmapped interpatient heterogeneity and disease complexity. We have utilized a single-cell cytometry-by-time-of-flight (cyTOF) based platform to precisely outline the effects of candidate drugs on human OA chondrocytes. OA chondrocytes harvested from patients undergoing total knee arthroplasty were treated with two drugs, an NF-kappaB pathway inhibitor, BMS-345541, and a chondroinductive small molecule, Kartogenin, that showed preclinical success in animal models for OA. cyTOF conducted with 30 metal isotope-labeled antibodies parsed the effects of the drugs on inflammatory, senescent, and chondroprogenitor populations. The NF-kappaB pathway inhibition decreased the expression of NF-kappaB, HIF2A and iNOS in multiple chondrocyte clusters and significantly depleted four p16ink4a expressing senescent populations including NOTCH1+STRO1+ chondroprogenitors. While Kartogenin also affected select p16ink4a expressing senescent clusters, there was a less discernible effect on chondroprogenitor populations. Overall, BMS-345541 elicited a uniform drug response in all patients while only a few responded to Kartogenin. These studies demonstrate that a single-cell cyTOF-based drug screening platform can provide insights into patient response assessment and their stratification.

  View details for DOI 10.1172/jci.insight.160702

  View details for PubMedID 36194485

• cPLA2 blockade attenuates S100A7-mediated breast tumorigenicity by inhibiting the immunosuppressive tumor microenvironment. Journal of experimental & clinical cancer research : CR Mishra, S., Charan, M., Shukla, R. K., Agarwal, P., Misri, S., Verma, A. K., Ahirwar, D. K., Siddiqui, J., Kaul, K., Sahu, N., Vyas, K., Garg, A. A., Khan, A., Miles, W. O., Song, J. W., Bhutani, N., Ganju, R. K. 2022; 41 (1): 54

  Abstract

  BACKGROUND: Molecular mechanisms underlying inflammation-associated breast tumor growth are poorly studied. S100A7, a pro-inflammatory molecule has been shown to enhance breast cancer growth and metastasis. However, the S100A7-mediated molecular mechanisms in enhancing tumor growth and metastasis are unclear.METHODS: Human breast cancer tissue and plasma samples were used to analyze the expression of S100A7, cPLA2, and PGE2. S100A7-overexpressing or downregulated human metastatic breast cancer cells were used to evaluate the S100A7-mediated downstream signaling mechanisms. Bi-transgenic mS100a7a15 overexpression, TNBC C3 (1)/Tag transgenic, and humanized patient-derived xenograft mouse models and cPLA2 inhibitor (AACOCF3) were used to investigate the role of S100A7/cPLA2/PGE2 signaling in tumor growth and metastasis. Additionally, CODEX, a highly advanced multiplexed imaging was employed to delineate the effects of S100A7/cPLA2 inhibition on the recruitment of various immune cells.RESULTS: In this study, we found that S100A7 and cPLA2 are highly expressed and correlate with decreased overall survival in breast cancer patients. Further mechanistic studies revealed that S100A7/RAGE signaling promotes the expression of cPLA2 to mediate its oncogenic effects. Pharmacological inhibition of cPLA2 suppressed S100A7-mediated tumor growth and metastasis in multiple pre-clinical models including transgenic and humanized patient-derived xenograft (PDX) mouse models. The attenuation of cPLA2 signaling reduced S100A7-mediated recruitment of immune-suppressive myeloid cells in the tumor microenvironment (TME). Interestingly, we discovered that the S100A7/cPLA2 axis enhances the immunosuppressive microenvironment by increasing prostaglandin E2 (PGE2). Furthermore, CO-Detection by indEXing (CODEX) imaging-based analyses revealed that cPLA2 inhibition increased the infiltration of activated and proliferating CD4+ and CD8+ T cells in the TME. In addition, CD163+ tumor associated-macrophages were positively associated with S100A7 and cPLA2 expression in malignant breast cancer patients.CONCLUSIONS: Our study provides new mechanistic insights on the cross-talk between S100A7/cPLA2 in enhancing breast tumor growth and metastasis by generating an immunosuppressive TME that inhibits the infiltration of cytotoxic T cells. Furthermore, our studies indicate that S100A7/cPLA2 could be used as novel prognostic marker and cPLA2 inhibitors as promising drugs against S100A7-overexpressing aggressive breast cancer.

  View details for DOI 10.1186/s13046-021-02221-0

  View details for PubMedID 35135586

• A Quick and Efficient Method for the Generation of Immunomodulatory MSC from Human iPSC. Tissue engineering. Part A Bruschi, M., Sahu, N., Singla, M., Grandi, F., Agarwal, P., Chu, C., Bhutani, N. 2021

  Abstract

  Mesenchymal stromal cells (MSC) have been widely investigated for their regenerative capacity, anti-inflammatory properties and beneficial immunomodulatory effects across multiple clinical indications. Nevertheless, their widespread clinical utilization is limited by the variability in MSC quality, impacted by donor age, metabolism and disease. Human induced pluripotent stem cells (hiPSC) generated from readily accessible donor tissues, are a promising source of stable and rejuvenated MSC but differentiation methods generally require prolonged culture and result in low frequencies of stable MSC. To overcome this limitation, we have optimized a quick and efficient method for hiPSC differentiation into foot-print free MSC (hiMSC) in this study. This method capitalizes on the synergistic action of growth factors Wnt3a and Activin A with BMP4, leading to an enrichment of MSC after only 4 days of treatment. These hiMSC demonstrate a significant upregulation of mesenchymal stromal markers (CD105+, CD90+, CD73 and CDH) comparable to bone marrow-derived MSC, with reduced expression of the pluripotency genes (Oct-4, c-Myc, Klf4 and Nanog) compared to hiPSC. Moreover, they show improved proliferation capacity in culture without inducing any teratoma formation in vivo. Osteogenesis, chondrogenesis and adipogenesis assays confirmed the ability of hiMSC to differentiate into the three different lineages. Secretome analyses showed cytokine profiles comparable to bone marrow-derived MSC. Encapsulated hiMSC in alginate beads co-cultured with osteoarthritic (OA) cartilage explants showed robust immunomodulation, with stimulation of cell growth and proteoglycan production in OA cartilage. Our quick and efficient protocol for derivation of hiMSC from hiPSC, and their encapsulation in microbeads therefore, presents a reliable and reproducible method to boost the clinical applications of MSC.

  View details for DOI 10.1089/ten.TEA.2021.0172

  View details for PubMedID 34693750

• Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture. Advanced materials (Deerfield Beach, Fla.) Indana, D., Agarwal, P., Bhutani, N., Chaudhuri, O. 2021: e2101966

  Abstract

  Organoids are lumen-containing multicellular structures that recapitulate key features of the organs, and are increasingly used in models of disease, drug testing, and regenerative medicine. Recent work has used 3D culture models to form organoids from human induced pluripotent stem cells (hiPSCs) in reconstituted basement membrane (rBM) matrices. However, rBM matrices offer little control over the microenvironment. More generally, the role of matrix viscoelasticity in directing lumen formation remains unknown. Here, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine-glycine-aspartate (RGD) ligand density are used to study hiPSC morphogenesis in 3Dculture. A phase diagram that shows how these properties control hiPSC morphogenesis is reported. Higher RGD density and fast stress relaxation promote hiPSC viability, proliferation, apicobasal polarization, and lumen formation, while slow stress relaxation at low RGD densities triggers hiPSC apoptosis. Notably, hiPSCs maintain pluripotency in alginate hydrogels for much longer times than is reported in rBM matrices. Lumen formation is regulated by actomyosin contractility and is accompanied by translocation of Yes-associated protein (YAP) from the nucleus to the cytoplasm. The results reveal matrix viscoelasticity as a potent factor regulating stem cell morphogenesis and provide new insights into how engineered biomaterials may be leveraged to build organoids.

  View details for DOI 10.1002/adma.202101966

  View details for PubMedID 34499389

• Preparation of Human Chondrocytes for Profiling Using Cytometry by Time-of-flight (cyTOF). Bio-protocol Grandi, F. C., Bhutani, N. 2021; 11 (14): e4086

  Abstract

  Single-cell technologies have allowed high-resolution profiling of tissues and thus a deeper understanding of tissue homeostasis and disease heterogeneity. Understanding this heterogeneity can be especially important for tailoring treatments in a patient-specific manner. Here, we detail methods for preparing human cartilage tissue for profiling via cytometry by time-of-flight (cyTOF). We have previously utilized this method to characterize several rare cell populations in cartilage, including cartilage-progenitor cells, inflammation-amplifying cells (Inf-A), and inflammation-dampening cells (Inf-D). Previous bio-protocols have focused on cyTOF staining of PBMCs. Therefore, here we detail the steps unique to the processing of human cartilage and chondrocytes. Briefly, cartilage tissue is digested to release individual chondrocytes, which can be expanded and manipulated in culture. These cells are then collected and fixed in preparation for cyTOF, followed by standard staining and analysis protocols.

  View details for DOI 10.21769/BioProtoc.4086

  View details for PubMedID 34395725

• Single Cell Omics for Musculoskeletal Research. Current osteoporosis reports Rai, M. F., Wu, C., Capellini, T. D., Guilak, F., Dicks, A. R., Muthuirulan, P., Grandi, F., Bhutani, N., Westendorf, J. J. 2021

  Abstract

  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.

  View details for DOI 10.1007/s11914-021-00662-2

  View details for PubMedID 33559841

• Mapping 5-Hydroxymethylcytosine (5hmC) Modifications in Skeletal Tissues Using High-Throughput Sequencing. Methods in molecular biology (Clifton, N.J.) Grandi, F. C., Bhutani, N. 2021; 2221: 101–8

  Abstract

  Cytosine modifications can alter the epigenetic landscape of a cell, affecting the binding of transcription factors, chromatin organizing complexes, and ultimately affecting gene expression and cell fate. 5-Hydroxymethylcytosine (5hmC) modifications are generated by the Ten-eleven-translocation (TET) family of enzymes, TET 1, 2, and 3, through the oxidation of methylated cytosines (5mC). The TET family is capable of further oxidizing 5hmC to 5fC and 5caC, leading to eventual DNA demethylation. However, 5hmC marks can also exist stably in DNA. Stable 5hmC is enriched in the gene bodies of activated genes in multiple tissues, as well as associated with regulatory regions such as enhancers. Alterations to 5hmC patterns have now been found in multiple diseases including osteoarthritis. Here, we describe a method to map 5hmC modifications by next-generation sequencing using a technique based on the selective modification and enrichment of the 5hmC mark. We additionally provide a bioinformatic analysis pipeline to interpret the resulting data.

  View details for DOI 10.1007/978-1-0716-0989-7_8

  View details for PubMedID 32979201

• Encapsulated Mesenchymal Stromal Cell Microbeads Promote Endogenous Regeneration of Osteoarthritic Cartilage Ex Vivo. Advanced healthcare materials Sahu, N. n., Agarwal, P. n., Grandi, F. n., Bruschi, M. n., Goodman, S. n., Amanatullah, D. n., Bhutani, N. n. 2021: e2002118

  Abstract

  The anti-inflammatory secretome of mesenchymal stromal cells (MSCs) is lucrative for the treatment of osteoarthritis (OA), a disease characterized by low-grade inflammation. However, the precise effects of the MSC secretome on patient-derived OA tissue is lacking. To investigate these effects, alginate encapsulated MSCs are co-cultured with patient-derived OA cartilage explants for 8 days. Proteoglycan distribution in OA cartilage explants examined by Safranin O staining is markedly improved when cultured with MSC microbeads as compared to control OA explants cultured alone. Total sulfated glycosaminoglycan (sGAG) content in OA explants is significantly increased upon co-culture with MSC microbeads on day 8. The sGAG released into the culture media is unchanged by the presence of MSC microbeads, suggesting de novo sGAG synthesis in OA explants. Co-culture with MSC microbeads increased the DNA content and Ki67+ cells in OA explants, indicating proliferation. An increase in secreted cytokines IL-10, HGF, and sFAS assessed by multiplex cytokine assay, increased TIMP1 levels, and reduction in percent apoptotic cells in OA explants is noted. Together, data demonstrates that paracrine factors secreted by alginate encapsulated MSCs microbeads in response to OA cartilage, create an anabolic, proliferative, and anti-apoptotic microenvironment inducing endogenous regeneration in clinically relevant, patient-derived OA cartilage.

  View details for DOI 10.1002/adhm.202002118

  View details for PubMedID 33434393

• TET1 Directs Chondrogenic Differentiation by Regulating SOX9 Dependent Activation of Col2a1 and Acan In Vitro. JBMR plus Smeriglio, P., Grandi, F. C., Taylor, S. E., Zalc, A., Bhutani, N. 2020; 4 (8): e10383

  Abstract

  Skeletal development is a tightly orchestrated process in which cartilage and bone differentiation are intricately intertwined. Recent studies have highlighted the contribution of epigenetic modifications and their writers to skeletal development. Methylated cytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the Ten-eleven-translocation (TET) enzymes leading to demethylation. We have previously demonstrated that 5hmC is stably accumulated on lineage-specific genes that are activated during in vitro chondrogenesis in the ATDC5 chondroprogenitors. Knockdown (KD) of Tet1 via short-hairpin RNAs blocked ATDC5 chondrogenic differentiation. Here, we aimed to provide the mechanistic basis for TET1 function during ATDC5 differentiation. Transcriptomic analysis of Tet1 KD cells demonstrated that 54% of downregulated genes were SOX9 targets, suggesting a role for TET1 in mediating activation of a subset of the SOX9 target genes. Using genome-wide mapping of 5hmC during ATDC5 differentiation, we found that 5hmC is preferentially accumulated at chondrocyte-specific class II binding sites for SOX9, as compared with the tissue-agnostic class I sites. Specifically, we find that SOX9 is unable to bind to Col2a1 and Acan after Tet1 KD, despite no changes in SOX9 levels. Finally, we compared this KD scenario with the genetic loss of TET1 in the growth plate using Tet1 -/- embryos, which are approximately 10% smaller than their WT counterparts. In E17.5 Tet1 -/- embryos, loss of SOX9 target gene expression is more modest than upon Tet1 KD in vitro. Overall, our data suggest a role for TET1-mediated 5hmC deposition in partly shaping an epigenome conducive for SOX9 function. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

  View details for DOI 10.1002/jbm4.10383

  View details for PubMedID 33134768

• Epigenetic Therapies for Osteoarthritis. Trends in pharmacological sciences Grandi, F. C., Bhutani, N. 2020

  Abstract

  Osteoarthritis (OA) is an age-associated disease characterized by chronic joint pain resulting from degradation of articular cartilage, inflammation of the synovial lining, and changes to the subchondral bone. Despite the wide prevalence, no FDA-approved disease-modifying drugs exist. Recent evidence has demonstrated that epigenetic dysregulation of multiple molecular pathways underlies OA pathogenesis, providing a new mechanistic and therapeutic axis with the advantage of targeting multiple deregulated pathways simultaneously. In this review, we focus on the epigenetic regulators that have been implicated in OA, their individual roles, and potential crosstalk. Finally, we discuss the pharmacological molecules that can modulate their activities and discuss the potential advantages and challenges associated with epigenome-based therapeutics for OA.

  View details for DOI 10.1016/j.tips.2020.05.008

  View details for PubMedID 32586653

• Single-cell mass cytometry reveals cross-talk between inflammation-dampening and inflammation-amplifying cells in osteoarthritic cartilage Science Advances Grandi, F. ., Baskar, R., Smeriglio, P., Murkherjee, S., Indelli, P., F. Amanatullah, D., Goodman, S., Chu, C., Bendall , S., Bhutani, N. 2020; 6 (11)

  View details for DOI 10.1126/sciadv.aay5352

• Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nature communications Sarkar, T. J., Quarta, M. n., Mukherjee, S. n., Colville, A. n., Paine, P. n., Doan, L. n., Tran, C. M., Chu, C. R., Horvath, S. n., Qi, L. S., Bhutani, N. n., Rando, T. A., Sebastiano, V. n. 2020; 11 (1): 1545

  Abstract

  Aging is characterized by a gradual loss of function occurring at the molecular, cellular, tissue and organismal levels. At the chromatin level, aging associates with progressive accumulation of epigenetic errors that eventually lead to aberrant gene regulation, stem cell exhaustion, senescence, and deregulated cell/tissue homeostasis. Nuclear reprogramming to pluripotency can revert both the age and the identity of any cell to that of an embryonic cell. Recent evidence shows that transient reprogramming can ameliorate age-associated hallmarks and extend lifespan in progeroid mice. However, it is unknown how this form of rejuvenation would apply to naturally aged human cells. Here we show that transient expression of nuclear reprogramming factors, mediated by expression of mRNAs, promotes a rapid and broad amelioration of cellular aging, including resetting of epigenetic clock, reduction of the inflammatory profile in chondrocytes, and restoration of youthful regenerative response to aged, human muscle stem cells, in each case without abolishing cellular identity.

  View details for DOI 10.1038/s41467-020-15174-3

  View details for PubMedID 32210226

• Platelet-Rich Plasma (PRP) From Older Males With Knee Osteoarthritis Depresses Chondrocyte Metabolism and Upregulates Inflammation JOURNAL OF ORTHOPAEDIC RESEARCH O'Donnell, C., Migliore, E., Grandi, F., Koltsov, J., Lingampalli, N., Cisar, C., Indelli, P. F., Sebastiano, V., Robinson, W. H., Bhutani, N., Chu, C. R. 2019; 37 (8): 1760–70

  View details for DOI 10.1002/jor.24322

  View details for Web of Science ID 000501249400010

• Platelet-Rich Plasma (PRP) from Older Males with Knee Osteoarthritis Depresses Chondrocyte Metabolism and Upregulates Inflammation. Journal of orthopaedic research : official publication of the Orthopaedic Research Society O'Donnell, C., Migliore, E., Grandi, F. C., Koltsov, J., Lingampalli, N., Cisar, C., Indelli, P. F., Sebastiano, V., Robinson, W. H., Bhutani, N., Chu, C. R. 2019

  Abstract

  There is intense clinical interest in the potential effects of platelet-rich plasma (PRP) for the treatment of osteoarthritis (OA). This study tested the hypotheses that (1) 'lower' levels of the inflammatory mediators (IM) interleukin-1-beta (IL-1beta) and tumor-necrosis-factor-alpha (TNF-alpha), and (2) 'higher' levels of the growth factors (GF) insulin-like-growth-factor-1 and transforming-growth-factor-beta-1 within leukocyte-poor PRP correlate with more favorable chondrocyte and macrophage responses in vitro. Samples were collected from ten 'healthy' young male (23-33 years old) human subjects (H-PRP) and nine older (62-85 years old) male patients with severe knee OA (OA-PRP). The samples were separated into groups of 'high' or 'low' levels of IM and GF based on multiplex cytokine and ELISA data. Three-dimensional (3D) alginate bead chondrocyte cultures and monocyte-derived macrophage cultures were treated with 10% PRP from donors in different groups. Gene expression was analyzed by qPCR. Contrary to our hypotheses, the effect of PRP on chondrocytes and macrophages was mainly influenced by the age and disease status of the PRP donor as opposed to the IM or GF groupings. While H-PRP showed similar effects on expression of chondrogenic markers (Col2a1 and Sox9) as the negative control group (p>0.05), OA-PRP decreased chondrocyte expression of Col2a1 and Sox-9 mRNA by 40% and 30%, respectively (Col2a1, p=0.015; Sox9, p=0.037). OA-PRP also upregulated TNF-alpha and MMP-9 (p<0.001) gene expression in macrophages while H-PRP did not. This data suggests that PRP from older individuals with OA contain factors that may suppress chondrocyte matrix synthesis and promote macrophage inflammation in vitro. This article is protected by copyright. All rights reserved.

  View details for PubMedID 31042308

• 4 Effect of trabecular metal on the elution of gentamicin from Palacos cement JOURNAL OF ORTHOPAEDIC RESEARCH Mooney, J. A., Manasherob, R., Smeriglio, P., Bhutani, N., Amanatullah, D. F. 2019; 37 (5): 1018–24

  View details for DOI 10.1002/jor.24274

  View details for Web of Science ID 000467453100003

• Effect of Trabecular Metal on the Elution of Gentamicin from Palacos Cement. Journal of orthopaedic research : official publication of the Orthopaedic Research Society Mooney, J. A., Manasherob, R., Smeriglio, P., Bhutani, N., Amanatullah, D. F. 2019

  Abstract

  Periprosthetic joint infections continues to be a common complication in total joint arthroplasty, resulting in significant morbidity, mortality and additional costs. Antibiotic loaded bone cement has profoundly reduced the incidence of infection and revision. Trabecular metal implants with an internal cemented interface may be customizable drug delivery devices with an ingrowth interface. Thirty-six acetabular implants were assembled in vitro, half with a trabecular metal shell and half without. The antibiotic loaded bone cement was prepared via three different mixing techniques and at two mixing times. Mixing time had a significant effect on the total amount of gentamicin eluted. The long mix protocol eluted up to 126% (p=0.001) more gentamicin than the short mix at four hours and 192% (p<0.001) more at seven days. The use of a trabecular metal shell had no significant effect at four hours (p>0.05) but significantly reduced total elution under certain mixing protocols at seven days. Mixing technique had no significant effect on elution at four hours. At seven days, the mechanical mixing system under vacuum eluted over 50% (p=0.031) more antibiotic than without a vacuum and nearly 60% (p=0.040) more antibiotic than hand mixing. The use of trabecular metal implants does not significantly inhibit the initial bulk elution of gentamicin. A possible optimization strategy to improve elution kinetics would be to use a long mixing time with a mechanical mixing system under vacuum. This article is protected by copyright. All rights reserved.

  View details for PubMedID 30839118

• Optimizing Clinical Use of Biologics in Orthopaedic Surgery: Consensus Recommendations From the 2018 AAOS/NIH U-13 Conference JOURNAL OF THE AMERICAN ACADEMY OF ORTHOPAEDIC SURGEONS Chu, C. R., Rodeo, S., Bhutani, N., Goodrich, L. R., Huard, J., Irrgang, J., LaPrade, R. F., Lattermann, C., Lu, Y., Mandelbaum, B., Mao, J., McIntyre, L., Mishra, A., Muschler, G. F., Piuzzi, N. S., Potter, H., Spindler, K., Tokish, J. M., Tuan, R., Zaslav, K., Maloney, W. 2019; 27 (2): E50–E63

  Abstract

  Concern that misinformation from direct-to-consumer marketing of largely unproven "biologic" treatments such as platelet-rich plasma and cell-based therapies may erode the public trust and the responsible investment needed to bring legitimate biological therapies to patients have resulted in calls to action from professional organizations and governing bodies. In response to substantial patient demand for biologic treatment of orthopaedic conditions, the American Academy of Orthopaedic Surgeons convened a collaborative symposium and established a consensus framework for improving and accelerating the clinical evaluation, use, and optimization of biologic therapies for musculoskeletal diseases. The economic and disease burden of musculoskeletal conditions is high. Of the various conditions discussed, knee osteoarthritis was identified as a "serious condition" associated with substantial and progressive morbidity and emerged as the condition with the most urgent need for clinical trial development. It was also recognized that stem cells have unique characteristics that are not met by minimally manipulated mixed cell preparations. The work group recommended that minimally manipulated cell products be referred to as cell therapy and that the untested and uncharacterized nature of these treatments be clearly communicated within the profession, to patients, and to the public. Minimum standards for product characterization and clinical research should also be followed. A framework for developing clinical trials related to knee OA was agreed upon. In addition to recommendations for development of high-quality multicenter clinical trials, another important recommendation was that physicians and institutions offering biologic therapies commit to establishing high-quality patient registries and biorepository-linked registries that can be used for postmarket surveillance and quality assessments.

  View details for DOI 10.5435/JAAOS-D-18-00305

  View details for Web of Science ID 000462411800001

  View details for PubMedID 30300216

  View details for PubMedCentralID PMC6314629

• Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing. Stem cells (Dayton, Ohio) Adkar, S. S., Wu, C., Willard, V. P., Dicks, A., Ettyreddy, A., Steward, N., Bhutani, N., Gersbach, C. A., Guilak, F. 2018

  Abstract

  The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here, we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells 2018.

  View details for PubMedID 30378731

• Highly Efficient Chondrogenic Differentiation of Human iPSCs and Purification via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing Adkar, S. S., Wu, C., Willard, V. P., Dicks, A., Ettyreddy, A., Steward, N., Bhutani, N., Gersbach, C. A., Guilak, F. CELL PRESS. 2018: 36

  View details for Web of Science ID 000435342200072

• Men and Women Differ in the Biochemical Composition of Platelet-Rich Plasma AMERICAN JOURNAL OF SPORTS MEDICINE Xiong, G., Lingampalli, N., Koltsov, J. B., Leung, L. L., Bhutani, N., Robinson, W. H., Chu, C. R. 2018; 46 (2): 409–19

  Abstract

  Autologous platelet-rich plasma (PRP) is widely used for a variety of clinical applications. However, clinical outcome studies have not consistently shown positive effects. The composition of PRP differs based on many factors. An improved understanding of factors influencing the composition of PRP is important for the optimization of PRP use.Age and sex influence the PRP composition in healthy patients.Controlled laboratory study.Blood from 39 healthy patients was collected at a standardized time and processed into leukocyte-poor PRP within 1 hour of collection using the same laboratory centrifuge protocol and frozen for later analysis. Eleven female and 10 male patients were "young" (aged 18-30 years), while 8 male and 10 female patients were "older" (aged 45-60 years). Thawed PRP samples were assessed for cytokine and growth factor levels using a multiplex assay and enzyme-linked immunosorbent assay. The platelet count and high-sensitivity C-reactive protein levels were measured. Two-way analysis of variance determined age- and sex-based differences.Platelet and high-sensitivity C-reactive protein concentrations were similar in PRP between the groups ( P = .234). Male patients had higher cytokine and growth factor levels in PRP compared with female patients for inflammatory cytokines such as interleukin-1 beta (IL-1β) (9.83 vs 7.71 pg/mL, respectively; P = .008) and tumor necrosis factor-alpha (TNF-α) (131.6 vs 110.5 pg/mL, respectively; P = .048); the anti-inflammatory IL-1 receptor antagonist protein (IRAP) (298.0 vs 218.0 pg/mL, respectively; P < .001); and growth factors such as fibroblast growth factor-basic (FGF-basic) (237.9 vs 194.0 pg/mL, respectively; P = .01), platelet-derived growth factor (PDGF-BB) (3296.2 vs 2579.3 pg/mL, respectively; P = .087), and transforming growth factor-beta 1 (TGF-β1) (118.8 vs 92.8 ng/mL, respectively; P = .002). Age- but not sex-related differences were observed for insulin-like growth factor-1 (IGF-1) ( P < .001). Age and sex interaction terms were not significant. While mean differences were significant, there was also substantial intragroup variability.This study in healthy patients shows differences in the composition of PRP between men and women, with sex being a greater factor than age. There was also proteomic variability within the groups. These data support a personalized approach to PRP treatment and highlight the need for a greater understanding of the relationships between proteomic factors in PRP and clinical outcomes.Variability in the proteomic profile of PRP may affect tissue and clinical responses to treatment. These data suggest that clinical studies should account for the composition of PRP used.

  View details for PubMedID 29211968

• Human iPSC-derived chondrocytes mimic juvenile chondrocyte function for the dual advantage of increased proliferation and resistance to IL-1 beta STEM CELL RESEARCH & THERAPY Lee, J., Smeriglio, P., Chu, C. R., Bhutani, N. 2017; 8: 244

  Abstract

  Induced pluripotent stem cells (iPSC) provide an unlimited patient-specific cell source for regenerative medicine. Adult cells have had limited success in cartilage repair, but juvenile chondrocytes (from donors younger than 13 years of age) have been identified to generate superior cartilage. With this perspective, the aim of these studies was to compare the human iPSC-derived chondrocytes (hiChondrocytes) to adult and juvenile chondrocytes and identify common molecular factors that govern their function.Phenotypic and functional characteristics of hiChondrocytes were compared to juvenile and adult chondrocytes. Analyses of global gene expression profiling, independent gene expression, and loss-of-function studies were utilized to test molecular factors having a regulatory effect on hiChondrocytes and juvenile chondrocyte function.Here, we report that the iPSC-derived chondrocytes mimic juvenile chondrocytes in faster cell proliferation and resistance to IL-1β compared to adult chondrocytes. Whole genome transcriptome analyses revealed unique ECM factors and immune response pathways to be enriched in both juvenile and iPSC-derived chondrocytes as compared to adult chondrocytes. Loss-of-function studies demonstrated that CD24, a cell surface receptor enriched in both juvenile chondrocytes and hiChondrocytes, is a regulatory factor in both faster proliferation and resistance to proinflammatory cues in these chondrocyte populations.Our studies identify that hiChondrocytes mimic juvenile chondrocytes for the dual advantage of faster proliferation and a reduced response to the inflammatory cytokine IL-1β. While developmental immaturity of iPSC-derived cells can be a challenge for tissues like muscle and brain, our studies demonstrate that it is advantageous for a tissue like cartilage that has limited regenerative ability in adulthood.

  View details for PubMedID 29096706

• Soluble Collagen VI treatment enhances mesenchymal stem cells expansion for engineering cartilage. Bioengineering & translational medicine Smeriglio, P., Lee, J., Bhutani, N. 2017; 2 (3): 278–84

  Abstract

  Bone Marrow-derived mesenchymal stem cells (BM-MSC) are an attractive source for cell-based therapies in cartilage injury owing to their efficient differentiation into chondrocytes and their immune-suppressive abilities. However, their clinical use is hampered by a scarcity of cells leading to compromised efficacy. While expansion of human MSC ex vivo can potentially overcome the scarcity of cells, current methods lead to a rapid loss of the stem cell properties. In this study, we report soluble Collagen VI (cartilage pericellular matrix component) as a potential biologic that can expand the MSC population while maintaining the stem cell phenotype as confirmed by expression of the stem cell markers CD105 and CD90. Short-term treatment with Collagen VI additionally retains the potential of MSC to differentiate into mature chondrocytes in pellet culture. Cartilage pellets generated from MSC treated with Collagen VI or control express comparable amounts of the chondrogenic markers Collagen II, Aggrecan and Sox9, and the extracellular glycosaminoglycans. Our observations confirm that the use of the endogenous and cartilage-specific factor Collagen VI is valuable for a rapid and efficient expansion of MSC for potential use in cartilage regeneration and osteoarthritis.

  View details for PubMedID 29313037

• The first international workshop on the epigenetics of osteoarthritis CONNECTIVE TISSUE RESEARCH Meulenbelt, I. M., Bhutani, N., den Hollander, W., Gay, S., Oppermann, U., Reynard, L. N., Skelton, A. J., Young, D. A., Beier, F., Loughlin, J. 2017; 58 (1): 37-48

  Abstract

  Osteoarthritis (OA) is a major clinical problem across the world, in part due to the lack of disease-modifying drugs resulting, to a significant degree, from our incomplete understanding of the underlying molecular mechanisms of the disease. Emerging evidence points to a role of epigenetics in the pathogenesis of OA, but research in this area is still in its early stages. In order to summarize current knowledge and to facilitate the potential coordination of future research activities, the first international workshop on the epigenetics of OA was held in Amsterdam in October 2015. Recent findings on DNA methylation and hydroxymethylation, histone modifications, noncoding RNAs, and other epigenetic mechanisms were presented and discussed. The workshop demonstrated the advantage of bringing together those working in this nascent field and highlights from the event are summarized in this report in the form of summaries from invited speakers and organizers.

  View details for DOI 10.3109/03008207.2016.1168409

  View details for Web of Science ID 000394521200005

• CD24 enrichment protects while its loss increases susceptibility of juvenile chondrocytes towards inflammation ARTHRITIS RESEARCH & THERAPY Lee, J., Smeriglio, P., Dragoo, J., Maloney, W. J., Bhutani, N. 2016; 18

  Abstract

  Diseases associated with human cartilage, including rheumatoid arthritis (RA) and osteoarthritis (OA) have manifested age, mechanical stresses and inflammation as the leading risk factors. Although inflammatory processes are known to be upregulated upon aging, we sought to gain a molecular understanding of how aging affects the tissue-specific response to inflammation. In this report, we explored the role of cluster of differentiation 24 (CD24) in regulating differential inflammatory responses in juvenile and adult human chondrocytes.Differential cell-surface CD24 expression was assessed in juvenile and adult chondrocytes along with human induced pluripotent stem cell (hiPSC)-derived neonatal chondrocytes through gene expression and fluorescence-activated cell sorting (FACS) analyses. Loss of function of CD24 was achieved through silencing in chondrocytes and the effects on the response to inflammatory cues were assessed through gene expression and NFκB activity.CD24 expression in chondrocytes caused a differential response to cytokine-induced inflammation, with the CD24(high) juvenile chondrocytes being resistant to IL-1ß treatment as compared to CD24(low) adult chondrocytes. CD24 protects from inflammatory response by reducing NFκB activation, as an acute loss of CD24 via silencing led to an increase in NFκB activation. Moreover, the loss of CD24 in chondrocytes subsequently increased inflammatory and catabolic gene expression both in the absence and presence of IL-1ß.We have identified CD24 as a novel regulator of inflammatory response in cartilage that is altered during development and aging and could potentially be therapeutic in RA and OA.

  View details for DOI 10.1186/s13075-016-1183-y

  View details for Web of Science ID 000390276900003

  View details for PubMedID 27955675

  View details for PubMedCentralID PMC5153697

• Identification of Human Juvenile Chondrocyte-Specific Factors that Stimulate Stem Cell Growth TISSUE ENGINEERING PART A Taylor, S. E., Lee, J., Smeriglio, P., Razzaque, A., Smith, R. L., Dragoo, J. L., Maloney, W. J., Bhutani, N. 2016; 22 (7-8): 645-653

  Abstract

  Although regeneration of human cartilage is inherently inefficient, age is an important risk factor for osteoarthritis. Recent reports have provided compelling evidence that juvenile chondrocytes (from donors below 13 years of age) are more efficient at generating articular cartilage as compared to adult chondrocytes. However, the molecular basis for such a superior regenerative capability is not understood. To identify the cell-intrinsic differences between juvenile and adult cartilage, we have systematically profiled global gene expression changes between a small cohort of human neonatal/juvenile and adult chondrocytes. No such study is available for human chondrocytes although young and old bovine and equine cartilage have been recently profiled. Our studies have identified and validated new factors enriched in juvenile chondrocytes as compared to adult chondrocytes including secreted extracellular matrix factors chordin-like 1 (CHRDL1) and microfibrillar-associated protein 4 (MFAP4). Network analyses identified cartilage development pathways, epithelial-mesenchymal transition, and innate immunity pathways to be overrepresented in juvenile-enriched genes. Finally, CHRDL1 was observed to aid the proliferation and survival of bone marrow-derived human mesenchymal stem cells (hMSC) while maintaining their stem cell potential. These studies, therefore, provide a mechanism for how young cartilage factors can potentially enhance stem cell function in cartilage repair.

  View details for DOI 10.1089/ten.tea.2015.0366

  View details for Web of Science ID 000374761600007

  View details for PubMedID 26955889

• Stable 5-Hydroxymethylcytosine (5hmC) Acquisition Marks Gene Activation During Chondrogenic Differentiation JOURNAL OF BONE AND MINERAL RESEARCH Taylor, S. E., Li, Y. H., Smeriglio, P., Rath, M., Wong, W. H., Bhutani, N. 2016; 31 (3): 524-534

  Abstract

  Regulation of gene expression changes during chondrogenic differentiation by DNA methylation and demethylation is little understood. Methylated cytosines (5mC) are oxidized by the ten-eleven-translocation (TET) proteins to 5-hydroxymethylcytosines (5hmC), 5-formylcytosines (5fC) and 5-carboxylcytosines (5caC) eventually leading to a replacement by unmethylated cytosines (C) i.e. DNA demethylation. Additionally, 5hmC is stable and acts as an epigenetic mark by itself. Here, we report that global changes in 5hmC mark chondrogenic differentiation in vivo and in vitro. Tibia anlagen and growth plate analyses during limb development at mouse embryonic days E 11.5, 13.5 and 17.5 showed dynamic changes in 5hmC levels in the differentiating chondrocytes. A similar increase in 5hmC levels was observed in the ATDC5 chondroprogenitor cell line accompanied by increased expression of the TET proteins during in vitro differentiation. Loss of TET1 in ATDC5 decreased 5hmC levels and impaired differentiation, demonstrating a functional role for TET1-mediated 5hmC dynamics in chondrogenic differentiation. Global analyses of the 5hmC-enriched sequences during early and late chondrogenic differentiation identified 5hmC distribution to be enriched in the regulatory regions of genes preceding the transcription start site (TSS) as well as in the gene bodies. Stable gains in 5hmC were observed in specific subsets of genes including genes associated with cartilage development and in chondrogenic lineage-specific genes. 5hmC gains in regulatory promoter and enhancer regions as well as in gene bodies were strongly associated with activated but not repressed genes, indicating a potential regulatory role for DNA hydroxymethylation in chondrogenic gene expression. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/jbmr.2711

  View details for Web of Science ID 000373596800006

  View details for PubMedID 26363184

• Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells FASEB JOURNAL Lee, J., Taylor, S. E., Smeriglio, P., Lai, J., Maloney, W. J., Yang, F., Bhutani, N. 2015; 29 (8): 3399-3410

  Abstract

  Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening.-Lee, J., Taylor, S. E. B., Smeriglio, P., Lai, J., Maloney, W. J., Yang, F., Bhutani, N. Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells.

  View details for DOI 10.1096/fj.14-269720

  View details for Web of Science ID 000358796900027

  View details for PubMedID 25911615

• Genome-Wide Mapping of DNA Hydroxymethylation in Osteoarthritic Chondrocytes ARTHRITIS & RHEUMATOLOGY Taylor, S. E., Li, Y. H., Wong, W. H., Bhutani, N. 2015; 67 (8): 2129-2140

  Abstract

  To examine the genome-wide distribution of hydroxymethylated cytosine (5hmC) in osteoarthritic (OA) and normal chondrocytes in order to investigate the effect on OA-specific gene expression.Cartilage was obtained from OA patients undergoing total knee arthroplasty or from control patients undergoing anterior cruciate ligament reconstruction. Genome-wide sequencing of 5hmC-enriched DNA was performed in a small cohort of normal and OA chondrocytes to identify differentially hydroxymethylated regions (DhMRs) in OA chondrocytes. Data from the genome-wide sequencing of 5hmC-enriched DNA were intersected with global OA gene expression data to define subsets of genes and pathways potentially affected by increased 5hmC levels in OA chondrocytes.A total of 70,591 DhMRs were identified in OA chondrocytes as compared to normal chondrocytes, 44,288 (63%) of which were increased in OA chondrocytes. The majority of DhMRs (66%) were gained in gene bodies. Increased DhMRs were observed in ∼50% of genes previously implicated in OA pathology including MMP3, LRP5, GDF5, and COL11A1. Furthermore, analyses of gene expression data revealed gene body gain of 5hmC appears to be preferentially associated with activated, but not repressed, genes in OA chondrocytes.This study provides the first genome-wide profiling of 5hmC distribution in OA chondrocytes. We had previously reported a global increase in 5hmC levels in OA chondrocytes. Gain of 5hmC in the gene body is found to be characteristic of activated genes in OA chondrocytes, highlighting the influence of 5hmC as an epigenetic mark in OA. In addition, this study identifies multiple OA-associated genes that are potentially regulated either singularly by gain of DNA hydroxymethylation or in combination with loss of DNA methylation.

  View details for DOI 10.1002/art.39179

  View details for Web of Science ID 000358609300018

  View details for PubMedCentralID PMC4519426

• Collagen VI Enhances Cartilage Tissue Generation by Stimulating Chondrocyte Proliferation. Tissue engineering. Part A Smeriglio, P., Dhulipala, L., Lai, J. H., Goodman, S. B., Dragoo, J. L., Smith, R. L., Maloney, W. J., Yang, F., Bhutani, N. 2015; 21 (3-4): 840-849

  Abstract

  Regeneration of human cartilage is inherently inefficient. Current cell-based approaches for cartilage repair, including autologous chondrocytes, are limited by the paucity of cells, associated donor site morbidity, and generation of functionally inferior fibrocartilage rather than articular cartilage. Upon investigating the role of collagen VI (Col VI), a major component of the chondrocyte pericellular matrix (PCM), we observe that soluble Col VI stimulates chondrocyte proliferation. Interestingly, both adult and osteoarthritis chondrocytes respond to soluble Col VI in a similar manner. The proliferative effect is, however, strictly due to the soluble Col VI as no proliferation is observed upon exposure of chondrocytes to immobilized Col VI. Upon short Col VI treatment in 2D monolayer culture, chondrocytes maintain high expression of characteristic chondrocyte markers like Col2a1, agc, and Sox9 whereas the expression of the fibrocartilage marker Collagen I (Col I) and of the hypertrophy marker Collagen X (Col X) is minimal. Additionally, Col VI-expanded chondrocytes show a similar potential to untreated chondrocytes in engineering cartilage in 3D biomimetic hydrogel constructs. Our study has, therefore, identified soluble Col VI as a biologic that can be useful for the expansion and utilization of scarce sources of chondrocytes, potentially for autologous chondrocyte implantation. Additionally, our results underscore the importance of further investigating the changes in chondrocyte PCM with age and disease and the subsequent effects on chondrocyte growth and function.

  View details for DOI 10.1089/ten.TEA.2014.0375

  View details for PubMedID 25257043

• Comparative potential of juvenile and adult human articular chondrocytes for cartilage tissue formation in three-dimensional biomimetic hydrogels. Tissue engineering. Part A Smeriglio, P., Lai, J. H., Dhulipala, L., Behn, A. W., Goodman, S. B., Smith, R. L., Maloney, W. J., Yang, F., Bhutani, N. 2015; 21 (1-2): 147-155

  Abstract

  Regeneration of human articular cartilage is inherently limited and extensive efforts have focused on engineering the cartilage tissue. Various cellular sources have been studied for cartilage tissue engineering including adult chondrocytes, as well as embryonic or adult stem cells. Juvenile chondrocytes (from donors below 13 years of age) have recently been reported to be a promising cell source for cartilage regeneration. Previous studies have compared the potential of adult and juvenile chondrocytes or adult and osteoarthritic (OA) chondrocytes. To comprehensively characterize the comparative potential of young, old and diseased chondrocytes, here we examined cartilage formation by juvenile, adult and OA chondrocytes in 3D biomimetic hydrogels composed of poly(ethylene glycol) and chondroitin sulfate. All three human articular chondrocytes were encapsulated in the 3D biomimetic hydrogels and cultured for 3 or 6 weeks to allow maturation and extracellular matrix formation. Outcomes were analyzed using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. After 3 and 6 weeks, juvenile chondrocytes showed a greater upregulation of chondrogenic gene expression than adult chondrocytes, while OA chondrocytes showed a downregulation. Aggrecan and type II collagen deposition and GAG accumulation were high for juvenile and adult chondrocytes but not for OA chondrocytes. Similar trend was observed in the compressive moduli of the cartilage constructs generated by the three different chondrocytes. In conclusion, the juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogels. The 3D culture model described here may also provide a useful tool to further study the molecular differences among chondrocytes from different stages, which can help elucidate the mechanisms for age-related decline in the intrinsic capacity for cartilage repair.

  View details for DOI 10.1089/ten.TEA.2014.0070

  View details for PubMedID 25054343

• 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation. Journal of visualized experiments : JoVE Smeriglio, P., Lai, J. H., Yang, F., Bhutani, N. 2015

  Abstract

  Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

  View details for DOI 10.3791/53085

  View details for PubMedID 26484414

  View details for PubMedCentralID PMC4692641

• A global increase in 5-hydroxymethylcytosine levels marks osteoarthritic chondrocytes. Arthritis & rheumatology (Hoboken, N.J.) Taylor, S. E., Smeriglio, P., Dhulipala, L., Rath, M., Bhutani, N. 2014; 66 (1): 90-100

  Abstract

  To investigate the role of the newly discovered epigenetic mark 5-hydroxymethylcytosine (5hmC) and its regulators in altered gene expression in osteoarthritis (OA).Cartilage was obtained from OA patients undergoing total knee arthroplasty and from control patients undergoing anterior cruciate ligament reconstruction. Global levels of 5hmC and 5-methylcytosine (5mC) were investigated using immunoblotting, enzyme-linked immunosorbent assays, and cellular staining. Gene expression changes were monitored by quantitative polymerase chain reaction (PCR) analysis. Levels of locus-specific 5hmC and 5mC at CpG sites in the matrix metalloproteinase 1 (MMP-1), MMP-3, ADAMTS-5, and hypoxanthine guanine phosphoribosyltransferase 1 (HPRT-1) promoters were quantified using a glucosylation and enzyme digestion-based method followed by quantitative PCR analysis. Global and locus-specific 5hmC levels and gene expression changes were monitored in normal chondrocytes stimulated with inflammatory cytokines to identify the effect of joint inflammation.A global 5-6-fold increase in 5hmC concomitant with a loss of TET1 was observed in human OA chondrocytes compared to normal chondrocytes. Enrichment of 5hmC was observed in promoters of enzymes critical to OA pathology, MMP-1 and MMP-3. Short-term treatment of normal chondrocytes with inflammatory cytokines induced a rapid decrease in TET1 expression but no global or locus-specific 5hmC enrichment.This study provides the first evidence of an epigenetic imbalance of the 5hmC homeostasis in OA leading to TET1 down-regulation and 5hmC accumulation. Our experiments identify 5hmC and its regulators as potential diagnostic and therapeutic targets in OA.

  View details for DOI 10.1002/art.38200

  View details for PubMedID 24449578

• A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells FASEB JOURNAL Bhutani, N., Decker, M. N., Brady, J. J., Bussat, R. T., Burns, D. M., Corbel, S. Y., Blau, H. M. 2013; 27 (3): 1107-1113

  Abstract

  Mechanistic insights into the reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) are limited, particularly for early acting molecular regulators. Here we use an acute loss of function approach to demonstrate that activation-induced deaminase (AID) activity is necessary for the initiation of reprogramming to iPSCs. While AID is well known for antibody diversification, it has also recently been shown to have a role in active DNA demethylation in reprogramming toward pluripotency and development. These findings suggested a potential role for AID in iPSC generation, yet, iPSC yield from AID-knockout mouse fibroblasts was similar to that of wild-type (WT) fibroblasts. We reasoned that an acute loss of AID function might reveal effects masked by compensatory mechanisms during development, as reported for other proteins. Accordingly, we induced an acute reduction (>50%) in AID levels using 4 different shRNAs and determined that reprogramming to iPSCs was significantly impaired by 79 ± 7%. The deaminase activity of AID was critical, as coexpression of WT but not a catalytic mutant AID rescued reprogramming. Notably, AID was required only during a 72-h time window at the onset of iPSC reprogramming. Our findings show a critical role for AID activity in the initiation of reprogramming to iPSCs.

  View details for DOI 10.1096/fj.12-222125

  View details for Web of Science ID 000315585200024

  View details for PubMedID 23212122

  View details for PubMedCentralID PMC3574289

• Cathepsins L and Z Are Critical in Degrading Polyglutamine-containing Proteins within Lysosomes JOURNAL OF BIOLOGICAL CHEMISTRY Bhutani, N., Piccirillo, R., Hourez, R., Venkatraman, P., Goldberg, A. L. 2012; 287 (21): 17471-17482

  Abstract

  In neurodegenerative diseases caused by extended polyglutamine (polyQ) sequences in proteins, aggregation-prone polyQ proteins accumulate in intraneuronal inclusions. PolyQ proteins can be degraded by lysosomes or proteasomes. Proteasomes are unable to hydrolyze polyQ repeat sequences, and during breakdown of polyQ proteins, they release polyQ repeat fragments for degradation by other cellular enzymes. This study was undertaken to identify the responsible proteases. Lysosomal extracts (unlike cytosolic enzymes) were found to rapidly hydrolyze polyQ sequences in peptides, proteins, or insoluble aggregates. Using specific inhibitors against lysosomal proteases, enzyme-deficient extracts, and pure cathepsins, we identified cathepsins L and Z as the lysosomal cysteine proteases that digest polyQ proteins and peptides. RNAi for cathepsins L and Z in different cell lines and adult mouse muscles confirmed that they are critical in degrading polyQ proteins (expanded huntingtin exon 1) but not other types of aggregation-prone proteins (e.g. mutant SOD1). Therefore, the activities of these two lysosomal cysteine proteases are important in host defense against toxic accumulation of polyQ proteins.

  View details for DOI 10.1074/jbc.M112.352781

  View details for Web of Science ID 000306373000047

  View details for PubMedID 22451661

• DNA Demethylation Dynamics CELL Bhutani, N., Burns, D. M., Blau, H. M. 2011; 146 (6): 866-872

  Abstract

  The discovery of cytosine hydroxymethylation (5hmC) suggested a simple means of demethylating DNA and activating genes. Further experiments, however, unearthed an unexpectedly complex process, entailing both passive and active mechanisms of DNA demethylation by the ten-eleven translocation (TET) and AID/APOBEC families of enzymes. The consensus emerging from these studies is that removal of cytosine methylation in mammalian cells can occur by DNA repair. These reports highlight that in certain contexts, DNA methylation is not fixed but dynamic, requiring continuous regulation.

  View details for DOI 10.1016/j.cell.2011.08.042

  View details for Web of Science ID 000295258100010

  View details for PubMedID 21925312

  View details for PubMedCentralID PMC3236603

• Reprogramming towards pluripotency requires AID-dependent DNA demethylation NATURE Bhutani, N., Brady, J. J., Damian, M., Sacco, A., Corbel, S. Y., Blau, H. M. 2010; 463 (7284): 1042-U57

  Abstract

  Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.

  View details for DOI 10.1038/nature08752

  View details for Web of Science ID 000275108400028

  View details for PubMedID 20027182

  View details for PubMedCentralID PMC2906123

• Nuclear reprogramming in heterokaryons is rapid, extensive, and bidirectional FASEB JOURNAL Palermo, A., Doyonnas, R., Bhutani, N., Pomerantz, J., Alkan, O., Blau, H. M. 2009; 23 (5): 1431-1440

  Abstract

  An understanding of nuclear reprogramming is fundamental to the use of cells in regenerative medicine. Due to technological obstacles, the time course and extent of reprogramming of cells following fusion has not been assessed to date. Here, we show that hundreds of genes are activated or repressed within hours of fusion of human keratinocytes and mouse muscle cells in heterokaryons, and extensive changes are observed within 4 days. This study was made possible by the development of a broadly applicable approach, species-specific transcriptome amplification (SSTA), which enables global resolution of transcripts derived from the nuclei of two species, even when the proportions of species-specific transcripts are highly skewed. Remarkably, either phenotype can be dominant; an excess of primary keratinocytes leads to activation of the keratinocyte program in muscle cells and the converse is true when muscle cells are in excess. We conclude that nuclear reprogramming in heterokaryons is rapid, extensive, bidirectional, and dictated by the balance of regulators contributed by the cell types.

  View details for DOI 10.1096/fj.08-122903

  View details for Web of Science ID 000266651700019

  View details for PubMedID 19141533

  View details for PubMedCentralID PMC2669427