DNA demethylation dynamics.

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.

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.

Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing.

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 2019;37:65-76.

The first international workshop on the epigenetics of osteoarthritis.

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.

Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells.

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.

Reprogramming towards pluripotency requires AID-dependent DNA demethylation.

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.

A critical role for AID in the initiation of reprogramming to induced pluripotent stem cells.

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.

Profiling joint tissues at single-cell resolution: advances and insights.

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.

Targeting an inflammation-amplifying cell population can attenuate osteoarthritis-associated pain.

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.

TET1 Regulates Skeletal Stem-Cell Mediated Cartilage Regeneration.

OBJECTIVE: Adult skeletal stem cells (SSCs) that 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. METHODS: We investigated the differences in the 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 sequencing, the transcriptomic differences between SSCs and bone cartilage stroma progenitor cells (BCSPs) were identified in Tet1+/+ mice and Tet1-/- mice. RESULTS: Loss of Tet1 skewed the SSC lineage tree by expanding the SSC pool and enhanced the chondrogenic potential of SSCs and BCSPs. Tet1 inhibition led to enhanced chondrogenesis in human SSCs and chondroprogenitors isolated from human cartilage. Importantly, TET1 inhibition in vivo in late stages of a mouse model of OA led to increased cartilage regeneration. Transcriptomic analyses of SSCs and BCSPs lacking Tet1 revealed pathway alterations in transforming growth factor ß signaling, melatonin degradation, and cartilage development-associated genes. Lastly, we report that use of the hormone melatonin can dampen inflammation and improve cartilage health. CONCLUSION: Although Tet1 is a broad epigenetic regulator, melatonin can mimic the inhibition ability of TET1 to enhance the chondrogenic ability of SSCs. Melatonin administration has the potential to be an attractive stem cell-based therapy for cartilage regeneration.

Lumen expansion is initially driven by apical actin polymerization followed by osmotic pressure in a human epiblast model.

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.

Laminin-associated integrins mediate Diffuse Intrinsic Pontine Glioma infiltration and therapy response within a neural assembloid model.

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.

Cathepsins L and Z are critical in degrading polyglutamine-containing proteins within lysosomes.

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.

A single-cell mass cytometry platform to map the effects of preclinical drugs on cartilage homeostasis.

No disease-modifying drug exists for osteoarthritis (OA). Despite success in animal models, candidate drugs continue to fail in clinical trials owing to the unmapped interpatient heterogeneity and disease complexity. We used a single-cell platform based on cytometry by time-of-flight (cyTOF) to precisely outline the effects of candidate drugs on human OA chondrocytes. OA chondrocytes harvested from patients undergoing total knee arthroplasty were treated with 2 drugs, an NF-κB 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 cell populations. The NF-κB pathway inhibition decreased the expression of p-NF-κB, HIF2A, and inducible NOS in multiple chondrocyte clusters and significantly depleted 4 p16ink4a-expressing senescent populations, including NOTCH1+STRO1+ chondroprogenitor cells. While kartogenin also affected select p16ink4a-expressing senescent clusters, there was a less discernible effect on chondroprogenitor cell 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 patient stratification.

A Quick and Efficient Method for the Generation of Immunomodulatory Mesenchymal Stromal Cell from Human Induced Pluripotent Stem Cell.

Mesenchymal stromal cells (MSCs) 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 (hiPSCs) 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 MSCs. To overcome this limitation, we have optimized a quick and efficient method for hiPSC differentiation into footprint-free MSCs (human induced MSCs [hiMSCs]) in this study. This method capitalizes on the synergistic action of growth factors Wnt3a and Activin A with bone morphogenetic protein-4 (BMP4), leading to an enrichment of MSC after only 4 days of treatment. These hiMSCs demonstrate a significant upregulation of mesenchymal stromal markers (CD105+, CD90+, CD73, and cadherin 11) compared with bone marrow-derived MSCs (bmMSCs), with reduced expression of the pluripotency genes (octamer-binding transcription factor [Oct-4], cellular myelocytomatosis oncogene [c-Myc], Klf4, and Nanog homebox [Nanog]) compared with 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 hiMSCs to differentiate into the three different lineages. Secretome analyses showed cytokine profiles compared with bmMSCs. Encapsulated hiMSCs in alginate beads cocultured 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 MSCs.

cPLA2 blockade attenuates S100A7-mediated breast tumorigenicity by inhibiting the immunosuppressive tumor microenvironment.

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.

Preparation of Human Chondrocytes for Profiling Using Cytometry by Time-of-flight (cyTOF).

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.

Mapping 5-Hydroxymethylcytosine (5hmC) Modifications in Skeletal Tissues Using High-Throughput Sequencing.

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.

Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture.

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 3D culture. 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.

Encapsulated Mesenchymal Stromal Cell Microbeads Promote Endogenous Regeneration of Osteoarthritic Cartilage Ex Vivo.

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.