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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. IMPACT STATEMENT: In this study, we present a novel, quick, and efficient method for the generation of mesenchymal stromal cells (MSCs) by differentiating human induced pluripotent stem cells in 4 days. The stably differentiatied and homogenous human induced MSCs (hiMSCs) are characterized for gene expression, differentiation, secretome, and immunomodulatory properties. Upon coculture with human osteoarthritic (OA) cartilage explants following encapsulation in microbeads, the hiMSC paracrine factors promote proteoglycan synthesis in the OA explants, demonstrating the potential of these hiMSCs to regenerate and repair cartilage.

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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 TRPV4 (transient receptor potential cation channel subfamily V member 4), which controls the phosphorylation of GSK3β (glycogen synthase kinase 3 beta), an enzyme with pleiotropic effects in osteoarthritis. But a dysfunctional TRPV4-GSK3β 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.

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.

Neuroinflammation after brain injury Traumatic brain injury affects millions of people every year and is a major cause of disability worldwide. Most of the maladaptive outcomes develop months or years later and are thought to be caused by secondary injuries that are indirect and long-term effects after the initial impact. Holden et al . found that secondary and chronic neuroinflammation and neurodegeneration are caused by the C1q molecule, a mediator of the complement pathway. C1q is responsible for chronic inflammation and secondary neuronal loss specifically in the cortico-thalamo-cortical circuit. Traumatic brain injury also leads to altered brain states that are caused by the C1q complement pathway. —PRS

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 (i) "lower" levels of the inflammatory mediators (IMs), interleukin-1β, and tumor necrosis factor α (TNF-α) and (ii) "higher" levels of the growth factors (GFs), insulin-like growth factor 1, and transforming growth factor β1 within leukocyte-poor PRP correlate with more favorable chondrocyte and macrophage responses in vitro. Samples were collected from 10 "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 enzyme-linked immunosorbent assay 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 quantitative polymerase chain reaction. 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 messenger RNA by 40% and 30%, respectively (Col2a1, p = 0.015; Sox9, p = 0.037). OA-PRP also upregulated TNF-α and matrix metallopeptidase 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. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1760-1770, 2019.

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. SUMMARY: This article summarizes talks given during a workshop on “Single Cell Omics” at the 2020 annual meeting of the Orthopedic Research Society. Studies that applied scRNA-seq, ATAC-seq, and CyTOF mass cytometry to cartilage development and osteoarthritis are reviewed. This body of work shows how these cutting-edge tools can advance our understanding of the cellular heterogeneity and trajectories of lineage specification during development and disease.

Single-cell 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.

Transgenerational transmission of epiphenotypes is poorly understood. Here we show that exposure of pregnant mouse F0 females to BPA results in obesity in the F2 progeny due to increased food intake and leptin resistance. This epiphenotype can be transmitted up to the F6 generation and disappears in F7. Analyses of chromatin accessibility in sperm of the F1-F6 generations reveals alterations in the binding of CTCF at two enhancers of the Fto gene in obese but not control animals that correlates with transmission of obesity. Deletion of the CTCF site in Fto results in mice that fail to become obese when exposed to BPA. These Fto enhancers show increased interactions in sperm of obese mice with the Irx3 and Irx5 genes, which are involved in the differentiation of appetite controlling AgRP/NPY neurons. Single-nucleus and immunofluorescence analyses in the arcuate nucleus of the hypothalamus suggest that exposure to BPA results in expansion of the number of orexigenic AgRP neurons. This expansion correlates with increased accessibility of the Fto proximal enhancer in radial glia-like neural stem cells (RG-NSCs), which give rise to AgRP/NPY neurons, and in mature oligodendrocytes. The results provide a molecular mechanism for transgenerational inheritance in mammals and suggest that both genetic and epigenetic alterations of Fto can lead to the same phenotypic outcomes.

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.

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.

In the mature cochlea, each inner hair cell (IHC) is innervated by multiple spiral ganglion neurons of type I (SGNI). SGNIs are morphologically and electro-physiologically diverse. Also, they differ in their susceptibility to noise insult. However, the molecular underpinnings of their identity and physiological differences remain poorly understood. In this study, we developed a novel triple transgenic mouse, which enabled the isolation of pure populations of SGNIs and the analysis of a 96-gene panel via single-cell qPCR. We found three distinct populations of Type I SGNs, which were marked by their exclusive expression of Lmx1a, Slc4a4, or Mfap4/Fzd2, respectively, at postnatal days P3, P8, and P12. Our data suggest that afferent SGN subtypes are established genetically before the onset of hearing and that the expression of key physiological markers, such as ion channels, is heterogeneous and may be underlying the heterogeneous firing proprieties of SGNIs.

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.

Long interspersed elements (LINEs), through both self-mobilization and trans-mobilization of short interspersed elements and processed pseudogenes, have made an indelible impact on the structure and function of the human genome. One consequence is the creation of new CpG islands (CGIs). In fact, more than half of all CGIs in the genome are associated with repetitive DNA, three-quarters of which are derived from retrotransposons. However, little is known about the epigenetic impact of newly inserted CGIs. We utilized a transgenic LINE-1 mouse model and tracked DNA methylation dynamics of individual germline insertions during mouse development. The retrotransposed GFP marker sequence, a strong CGI, is hypomethylated in male germ cells but hypermethylated in somatic tissues, regardless of genomic location. The GFP marker is similarly methylated when delivered into the genome via the Sleeping Beauty DNA transposon, suggesting that the observed methylation pattern may be independent of the mode of insertion. Comparative analyses between insertion- and non-insertion-containing alleles further reveal a graded influence of the retrotransposed CGI on flanking CpG sites, a phenomenon that we described as “sloping shores.” Computational analyses of human and mouse methylomic data at single-base resolution confirm that sloping shores are universal for hypomethylated CGIs in sperm and somatic tissues. Additionally, the slope of a hypomethylated CGI can be affected by closely positioned CGI neighbors. Finally, by tracing sloping shore dynamics through embryonic and germ cell reprogramming, we found evidence of bookmarking, a mechanism that likely determines which CGIs will be eventually hyper- or hypomethylated.

This study defines a single-cell proteomic atlas of human OA cartilage to reveal rare subpopulations that stratify patients., Aging or injury leads to degradation of the cartilage matrix and the development of osteoarthritis (OA). Because of a paucity of single-cell studies of OA cartilage, little is known about the interpatient variability in its cellular composition and, more importantly, about the cell subpopulations that drive the disease. Here, we profiled healthy and OA cartilage samples using mass cytometry to establish a single-cell atlas, revealing distinct chondrocyte progenitor and inflammation-modulating subpopulations. These rare populations include an inflammation-amplifying (Inf-A) population, marked by interleukin-1 receptor 1 and tumor necrosis factor receptor II, whose inhibition decreased inflammation, and an inflammation-dampening (Inf-D) population, marked by CD24, which is resistant to inflammation. We devised a pharmacological strategy targeting Inf-A and Inf-D cells that significantly decreased inflammation in OA chondrocytes. Using our atlas, we stratified patients with OA in three groups that are distinguished by the relative proportions of inflammatory to regenerative cells, making it possible to devise precision therapeutic approaches.

The PIWI-interacting RNA (piRNA) pathway is essential for retrotransposon silencing. In piRNA-deficient mice, L1-overexpressing male germ cells exhibit excessive DNA damage and meiotic defects. It remains unknown whether L1 expression simply highlights piRNA deficiency or actually drives the germ-cell demise. Specifically, the sheer abundance of genomic L1 copies prevents reliable quantification of new insertions. Here, we developed a codon-optimized L1 transgene that is controlled by an endogenous mouse L1 promoter. Importantly, DNA methylation dynamics of a single-copy transgene were indistinguishable from those of endogenous L1s. Analysis of Mov10l1-/- testes established that de novo methylation of the L1 transgene required the intact piRNA pathway. Consistent with loss of DNA methylation and programmed reduction of H3K9me2 at meiotic onset, the transgene showed 1,400-fold increase in RNA expression and consequently 70-fold increase in retrotransposition in postnatal day 14 Mov10l1-/- germ cells compared with the wild-type. Analysis of adult Mov10l1-/- germ-cell fractions indicated a stage-specific increase of retrotransposition in the early meiotic prophase. However, extrapolation of the transgene data to endogenous L1s suggests that it is unlikely insertional mutagenesis alone accounts for the Mov10l1-/- phenotype. Indeed, pharmacological inhibition of reverse transcription did not rescue the meiotic defect. Cumulatively, these results establish the occurrence of productive L1 mobilization in the absence of an intact piRNA pathway but leave open the possibility of processes preceding L1 integration in triggering meiotic checkpoints and germ-cell death. Additionally, our data suggest that many heritable L1 insertions originate from individuals with partially compromised piRNA defense.

Interspersed and tandem repeat sequences comprise the bulk of mammalian genomes. Interspersed repeats result from successive replication by transposable elements, such as Alu and long interspersed element type 1 (L1). Microsatellites are tandem repeats of 1–6 base pairs, among which poly(A) microsatellites are the most abundant in the human genome. The rise and fall of a microsatellite has been depicted as a life cycle. Previous studies have demonstrated that Alu and L1 insertions are a major source of A-rich microsatellites owing to the concurrent formation of a poly(A) DNA tract at the 3′-end of each insertion. The fate of such poly(A) tracts has been studied by surveying the length distribution of genomic resident Alu and L1 insertions. However, these cross-sectional studies provide no information about the tempo of mutation immediately after birth. In this study, de novo L1 insertions were created using a transgenic L1 mouse model and traced through generations to investigate the early life of poly(A) microsatellites. High frequencies of intra-individual and intergenerational shortening were observed for long poly(A) tracts, creating somatic and germline mosaicism at the insertion site, whereas little variation was observed for short poly(A) alleles. As poly(A) microsatellites are the major intrinsic signal for nucleosome positioning, their remarkable abundance and variability make them a significant source of epigenetic variation. Thus, the birth of poly(A) microsatellites from retrotransposons and the subsequent rapid and variable shortening represent a new way with which retrotransposons can modify the genetic and epigenetic architecture of our genome.

Epidermolysis Bullosa (EB) encompasses a spectrum of mechanobullous disorders caused by rare mutations that result in structural weakening of the skin and mucous membranes. While gene mutated and types of mutations present are broadly predictive of the range of disease to be expected, a remarkable amount of phenotypic variability remains unaccounted for in all but the most deleterious cases. This unexplained variance raises the possibility of genetic modifier effects. We tested this hypothesis using a mouse model that recapitulates a non-Herlitz form of junctional EB (JEB) owing to the hypomorphic jeb allele of laminin gamma 2 (Lamc2). By varying normally asymptomatic background genetics, we document the potent impact of genetic modifiers on the strength of dermal-epidermal adhesion and on the clinical severity of JEB in the context of the Lamc2jeb mutation. Through an unbiased genetic approach involving a combination of QTL mapping and positional cloning, we demonstrate that Col17a1 is a strong genetic modifier of the non-Herlitz JEB that develops in Lamc2jeb mice. This modifier is defined by variations in 1–3 neighboring amino acids in the non-collagenous 4 domain of the collagen XVII protein. These allelic variants alter the strength of dermal-epidermal adhesion in the context of the Lamc2jeb mutation and, consequentially, broadly impact the clinical severity of JEB. Overall the results provide an explanation for how normally innocuous allelic variants can act epistatically with a disease causing mutation to impact the severity of a rare, heritable mechanobullous disorder., Author Summary Epidermolysis bullosa (EB) is a group of rare genetic Mendelian disorders that result in mechanical fragility of the skin and mucosal membranes. Junctional EB is a subset caused by mutations that result in cleavage of the dermal-epidermal junction. All forms of EB demonstrate substantial variability in their clinical phenotype that is not readily explained. The possibility of genetic modifiers as the cause of this variability has been difficult to address in humans. We apply a mouse model carrying a hypomorphic allele of the laminin gamma 2 (Lamc2) gene to address the possibility of genetic modifiers of JEB. We document the potent impact of differing genetic backgrounds on multiple facets of the JEB syndrome expressed in these mice and show that three neighboring amino acid changes within the non-collagenous domain 4 of the collagen XVII protein strongly modify their disease. The study provides a molecular explanation of how a primary mutation that weakens one component of the cutaneous basement membrane is influenced by normally innocuous allelic variants of another component to affect strength of dermal-epidermal adhesion and consequently, the severity of JEB. This approach may guide the genetic prognosis and diagnosis of human EB disorders.

The human genome is laden with both non-LTR (long-terminal repeat) retrotransposons and microsatellite repeats. Both types of sequences are able to, either actively or passively, mutagenize the genomes of human individuals and are therefore poised to dynamically alter the human genomic landscape across generations. Non-LTR retrotransposons, such as L1 and Alu, are a major source of new microsatellites, which are born both concurrently and subsequently to L1 and Alu integration into the genome. Likewise, the mutation dynamics of microsatellite repeats have a direct impact on the fitness of their non-LTR retrotransposon parent owing to microsatellite expansion and contraction. This review explores the interactions and dynamics between non-LTR retrotransposons and microsatellites in the context of genomic variation and evolution.