Skewing of the population balance of lymphoid and myeloid cells by secreted and intracellular osteopontin.

The balance of myeloid populations and lymphoid populations must be well controlled. Here we found that osteopontin (OPN) skewed this balance during pathogenic conditions such as infection and autoimmunity. Notably, two isoforms of OPN exerted distinct effects in shifting this balance through cell-type-specific regulation of apoptosis. Intracellular OPN (iOPN) diminished the population size of myeloid progenitor cells and myeloid cells, and secreted OPN (sOPN) increase the population size of lymphoid cells. The total effect of OPN on skewing the leukocyte population balance was observed as host sensitivity to early systemic infection with Candida albicans and T cell-mediated colitis. Our study suggests previously unknown detrimental roles for two OPN isoforms in causing the imbalance of leukocyte populations.

Conformational control of Cas9 by CRISPR hybrid RNA-DNA guides mitigates off-target activity in T cells.

The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2'-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.

Single-Cell RNA Sequencing Identifies Yes-Associated Protein 1-Dependent Hepatic Mesothelial Progenitors in Fibrolamellar Carcinoma.

Fibrolamellar carcinoma (FLC) is characterized by in-frame fusion of DnaJ heat shock protein family (Hsp40) member B1 (DNAJB1) with protein kinase cAMP-activated catalytic subunit α (PRKACA) and by dense desmoplasia. Surgery is the only effective treatment because mechanisms supporting tumor survival are unknown. We used single-cell RNA sequencing to characterize a patient-derived FLC xenograft model and identify therapeutic targets. Human FLC cells segregated into four discrete clusters that all expressed the oncogene Yes-associated protein 1 (YAP1). The two communities most enriched with cells coexpressing FLC markers [CD68, A-kinase anchoring protein 12 (AKAP12), cytokeratin 7, epithelial cell adhesion molecule (EPCAM), and carbamoyl palmitate synthase-1] also had the most cells expressing YAP1 and its proproliferative target genes (AREG and CCND1), suggesting these were proliferative FLC cell clusters. The other two clusters were enriched with cells expressing profibrotic YAP1 target genes, ACTA2, ELN, and COL1A1, indicating these were fibrogenic FLC cells. All clusters expressed the YAP1 target gene and mesothelial progenitor marker mesothelin, and many mesothelin-positive cells coexpressed albumin. Trajectory analysis predicted that the four FLC communities were derived from a single cell type transitioning among phenotypic states. After establishing a novel FLC cell line that harbored the DNAJB1-PRKACA fusion, YAP1 was inhibited, which significantly reduced expression of known YAP1 target genes as well as cell growth and migration. Thus, both FLC epithelial and stromal cells appear to arise from DNAJB1-PRKACA fusion in a YAP1-dependent liver mesothelial progenitor, identifying YAP1 as a target for FLC therapy.

Development of intestinal organoids as tissue surrogates: cell composition and the epigenetic control of differentiation.

Intestinal organoids are multicellular crypt-like structures that can be derived from adult intestinal stem cells (ISCs), embryonic stem cells (ESCs) or induced pluripotent stem cells (IPSCs). Here we show that intestinal organoids generated from mouse ESCs were enriched in ISCs and early progenitors. Treatment of these organoids with a γ-secretase inhibitor increased Math1 and decreased Hes1 expression, indicating Notch signaling regulates ISC differentiation in these organoids. Lgr5 and Tert positive ISCs constituted approximately 10% and 20% of the organoids. As found in native tissue, Lgr5 and Tert expressing cells resolved into two discreet populations, which were stable over time. Intestinal organoids derived from cancer-prone Apc(Min/+) mice showed similar numbers of ISCs, but had reduced Math1 expression, indicating a suppressed secretory cell differentiation potential (as found in intestinal tissue). Apc(Min/+) organoids were used to screen epigenetically active compounds for those that increased Math1 expression and organoid differentiation (including HDAC inhibitors, Sirtuin (SIRT) modulators and methyltransferase inhibitors). Broad-spectrum HDAC inhibitors increased both Math1 and Muc2 expression, indicating an ability to promote the suppressed secretory cell differentiation pathway. Other epigenetic compounds had a diverse impact on cell differentiation, with a strong negative correlation between those that activated the secretory marker Muc2 and those that activated the absorptive cell marker Fabp2. These data show that ESC-derived intestinal organoids can be derived in large numbers, contain distinct ISC types and can be used to screen for agents that promote cell differentiation through different lineage pathways.

Efficient differentiation of human iPSC-derived mesenchymal stem cells to chondroprogenitor cells.

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell-based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC-derived mesenchymal-like progenitor population. We found the direct plating of undifferentiated iPSCs into high-density micromass cultures in the presence of BMP-2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP-2 treatment of iPSC-derived MSC-like (iPSC-MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage-specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell-based approaches to repair joint cartilage damage.

SCLD: a Stem Cell Lineage Database for the annotation of cell types and developmental lineages.

Stem cell biology has experienced explosive growth over the past decade as researchers attempt to generate therapeutically relevant cell types in the laboratory. Recapitulation of endogenous developmental trajectories is a dominant paradigm in the design of directed differentiation protocols, and attempts to guide stem cell differentiation are often based explicitly on knowledge of in vivo development. Therefore, when designing protocols, stem cell biologists rely heavily upon information including (i) cell type-specific gene expression profiles, (ii) anatomical and developmental relationships between cells and tissues and (iii) signals important for progression from progenitors to target cell types. Here, we present the Stem Cell Lineage Database (SCLD) (http://scld.mcb.uconn.edu) that aims to unify this information into a single resource where users can easily store and access information about cell type gene expression, cell lineage maps and stem cell differentiation protocols for both human and mouse stem cells and endogenous developmental lineages. By establishing the SCLD, we provide scientists with a centralized location to organize access and share data, dispute and resolve contentious relationships between cell types and within lineages, uncover discriminating cell type marker panels and design directed differentiation protocols.

Patient-Family and Provider Partnerships in the Management of Pediatric Uncomplicated Appendicitis.

In recent years, studies have demonstrated non-operative management with antibiotics alone to be a safe and effective treatment option for children with uncomplicated acute appendicitis. Shared decision-making is critical in the treatment of uncomplicated appendicitis due to the markedly different risks and benefits associated with surgery and non-operative management. In this report, we discuss the importance of shared decision-making in surgery using a case of uncomplicated appendicitis as an example. We present both the patient-family and provider perspectives on evaluating and deciding between operative and non-operative management and discuss the value of shared decision-making in the unique setting of an acute pathologic process with surgical and medical treatment options.

Genetic and epigenetic signatures associated with plasma oxytocin levels in children and adolescents with autism spectrum disorder.

Oxytocin (OT), the brain's most abundant neuropeptide, plays an important role in social salience and motivation. Clinical trials of the efficacy of OT in autism spectrum disorder (ASD) have reported mixed results due in part to ASD's complex etiology. We investigated whether genetic and epigenetic variation contribute to variable endogenous OT levels that modulate sensitivity to OT therapy. To carry out this analysis, we integrated genome-wide profiles of DNA-methylation, transcriptional activity, and genetic variation with plasma OT levels in 290 participants with ASD enrolled in a randomized controlled trial of OT. Our analysis identified genetic variants with novel association with plasma OT, several of which reside in known ASD risk genes. We also show subtle but statistically significant association of plasma OT levels with peripheral transcriptional activity and DNA-methylation profiles across several annotated gene sets. These findings broaden our understanding of the effects of the peripheral oxytocin system and provide novel genetic candidates for future studies to decode the complex etiology of ASD and its interaction with OT signaling and OT-based interventions. LAY SUMMARY: Oxytocin (OT) is an abundant chemical produced by neurons that plays an important role in social interaction and motivation. We investigated whether genetic and epigenetic factors contribute to variable OT levels in the blood. To this, we integrated genetic, gene expression, and non-DNA regulated (epigenetic) signatures with blood OT levels in 290 participants with autism enrolled in an OT clinical trial. We identified genetic association with plasma OT, several of which reside in known autism risk genes. We also show statistically significant association of plasma OT levels with gene expression and epigenetic across several gene pathways. These findings broaden our understanding of the factors that influence OT levels in the blood for future studies to decode the complex presentation of autism and its interaction with OT and OT-based treatment.

Mechanism of amylin fibrillization enhancement by heparin.

We characterized the interaction of amylin with heparin fragments of defined length, which model the glycosaminoglycan chains associated with amyloid deposits found in type 2 diabetes. Binding of heparin fragments to the positively charged N-terminal half of monomeric amylin depends on the concentration of negatively charged saccharides but is independent of oligosaccharide length. By contrast, amylin fibrillogenesis has a sigmoidal dependence on heparin fragment length, with an enhancement observed for oligosaccharides longer than four monomers and a leveling off of effects beyond 12 monomers. The length dependence suggests that the negatively charged helical structure of heparin electrostatically complements the positively charged surface of the fibrillar amylin cross-ß structure. Fluorescence resonance energy transfer and total internal reflection fluorescence microscopy experiments indicate that heparin associates with amylin fibrils, rather than enhancing fibrillogenesis catalytically. Short heparin fragments containing two- or eight-saccharide monomers protect against amylin cytotoxicity toward a MIN6 mouse cell model of pancreatic ß-cells.

Mitochondrial genetic background in Ghanaian patients with primary open-angle glaucoma.

PURPOSE: Prevalence rates for primary open-angle glaucoma (POAG) are significantly higher in Africans than in European or Asians. It has been reported recently that mitochondrial DNA (mtDNA) lineages of African origin, excluding L2, conferred susceptibility to POAG in Saudi Arabia. This prompted us to test the role of mtDNA haplogroups in the incidence of POAG in the Ghanaian population who has a high frequency of L2 lineages. METHODS: DNA was extracted from two independent cohorts of clinically diagnosed POAG patients (n=373) and healthy controls (n=451). All patients and controls were from Accra and Tema (the southern region of Ghana). The hypervariable region-I (HVS-I) and coding regions comprising mtDNA haplogroup diagnostic polymorphisms were polymerase chain reaction (PCR) amplified and sequenced in all patients and controls and the haplotypes obtained were assorted into haplogroups and their frequencies compared between cohorts. RESULTS: No statistically significant differences were found in mtDNA haplogroup frequencies between POAG patients and matched controls in this cohort for the various mtDNA haplogroups tested. CONCLUSIONS: In this Ghanaian cohort, mtDNA haplogroups do not seem to confer susceptibility to POAG.

Intestinal lineage commitment of embryonic stem cells.

Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.

Synovial cell cross-talk with cartilage plays a major role in the pathogenesis of osteoarthritis.

We elucidated the molecular cross-talk between cartilage and synovium in osteoarthritis, the most widespread arthritis in the world, using the powerful tool of single-cell RNA-sequencing. Multiple cell types were identified based on profiling of 10,640 synoviocytes and 26,192 chondrocytes: 12 distinct synovial cell types and 7 distinct articular chondrocyte phenotypes from matched tissues. Intact cartilage was enriched for homeostatic and hypertrophic chondrocytes, while damaged cartilage was enriched for prefibro- and fibro-, regulatory, reparative and prehypertrophic chondrocytes. A total of 61 cytokines and growth factors were predicted to regulate the 7 chondrocyte cell phenotypes. Based on production by > 1% of cells, 55% of the cytokines were produced by synovial cells (39% exclusive to synoviocytes and not expressed by chondrocytes) and their presence in osteoarthritic synovial fluid confirmed. The synoviocytes producing IL-1beta (a classic pathogenic cytokine in osteoarthritis), mainly inflammatory macrophages and dendritic cells, were characterized by co-expression of surface proteins corresponding to HLA-DQA1, HLA-DQA2, OLR1 or TLR2. Strategies to deplete these pathogenic intra-articular cell subpopulations could be a therapeutic option for human osteoarthritis.

Meteorin-like facilitates skeletal muscle repair through a Stat3/IGF-1 mechanism.

The immune system plays a multifunctional role throughout the regenerative process, regulating both pro-/anti-inflammatory phases and progenitor cell function. In the present study, we identify the myokine/cytokine Meteorin-like (Metrnl) as a critical regulator of muscle regeneration. Mice genetically lacking Metrnl have impaired muscle regeneration associated with a reduction in immune cell infiltration and an inability to transition towards an anti-inflammatory phenotype. Isochronic parabiosis, joining wild-type and whole-body Metrnl knock-out (KO) mice, returns Metrnl expression in the injured muscle and improves muscle repair, providing supportive evidence for Metrnl secretion from infiltrating immune cells. Macrophage-specific Metrnl KO mice are also deficient in muscle repair. During muscle regeneration, Metrnl works, in part, through Stat3 activation in macrophages, resulting in differentiation to an anti-inflammatory phenotype. With regard to myogenesis, Metrnl induces macrophage-dependent insulin-like growth factor 1 production, which has a direct effect on primary muscle satellite cell proliferation. Perturbations in this pathway inhibit efficacy of Metrnl in the regenerative process. Together, these studies identify Metrnl as an important regulator of muscle regeneration and a potential therapeutic target to enhance tissue repair.

Author Correction: Meteorin-like facilitates skeletal muscle repair through a Stat3/IGF-1 mechanism.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Erythromyeloid progenitors give rise to a population of osteoclasts that contribute to bone homeostasis and repair.

Osteoclasts are multinucleated cells of the monocyte/macrophage lineage that degrade bone. Here, we used lineage tracing studies-labelling cells expressing Cx3cr1, Csf1r or Flt3-to identify the precursors of osteoclasts in mice. We identified an erythromyeloid progenitor (EMP)-derived osteoclast precursor population. Yolk-sac macrophages of EMP origin produced neonatal osteoclasts that can create a space for postnatal bone marrow haematopoiesis. Furthermore, EMPs gave rise to long-lasting osteoclast precursors that contributed to postnatal bone remodelling in both physiological and pathological settings. Our single-cell RNA-sequencing data showed that EMP-derived osteoclast precursors arose independently of the haematopoietic stem cell (HSC) lineage and the data from fate tracking of EMP and HSC lineages indicated the possibility of cell-cell fusion between these two lineages. Cx3cr1+ yolk-sac macrophage descendants resided in the adult spleen, and parabiosis experiments showed that these cells migrated through the bloodstream to the remodelled bone after injury.

No association between OPA1 polymorphisms and primary open-angle glaucoma in three different populations.

PURPOSE: To investigate whether recently described polymorphisms in the optic atrophy 1 gene (OPA1) are associated with primary open-angle glaucoma (POAG) with elevated intraocular pressure in the Caucasian, African-American, and Ghanaian (West African) populations. METHODS: POAG was defined as the presence of glaucomatous optic nerve damage, associated visual field loss, and elevated intraocular pressure (>21 mm of mercury in both eyes). We used TaqMan allelic discrimination assays to genotype two single nucleotide polymorphisms (SNPs, rs10451941 and rs166850) in OPA1 in the Caucasian (279 cases, 227 controls), African American (193 cases, 97 controls), and Ghanaian (170 cases, 138 controls) populations. Allele, genotype, and haplotype frequencies were compared between the cases and controls from each population. RESULTS: There was no significant difference in OPA1 allele or genotype frequencies between POAG patients and controls at the rs10451941 and rs166850 SNPs in any population (p>0.05). Haplotype analysis also failed to demonstrate a significant association with POAG. The age-of-onset distribution in the Caucasian POAG patients was independent from genotypes at rs10451941. CONCLUSIONS: There was no association between two previously implicated OPA1 polymorphisms and a POAG phenotype that includes elevated intraocular pressure. This represents the first association analysis of OPA1 in high tension glaucoma in the African American and Ghanaian populations and is the largest study to date on the investigation of the potential association between OPA1 and POAG with elevated intraocular pressure. OPA1 association with POAG may be limited to patients with normal tension glaucoma in these populations.

Regeneration of Articular Cartilage by Human ESC-Derived Mesenchymal Progenitors Treated Sequentially with BMP-2 and Wnt5a.

The success of cell-based therapies to restore joint cartilage requires an optimal source of reparative progenitor cells and tight control of their differentiation into a permanent cartilage phenotype. Bone morphogenetic protein 2 (BMP-2) has been extensively shown to promote mesenchymal cell differentiation into chondrocytes in vitro and in vivo. Conversely, developmental studies have demonstrated decreased chondrocyte maturation by Wingless-Type MMTV Integration Site Family, Member 5A (Wnt5a). Thus, we hypothesized that treatment of human embryonic stem cell (hESC)-derived chondroprogenitors with BMP-2 followed by Wnt5a may control the maturational progression of these cells into a hyaline-like chondrocyte phenotype. We examined the effects of sustained exposure of hESC-derived mesenchymal-like progenitors to recombinant Wnt5a or BMP-2 in vitro. Our data indicate that BMP-2 promoted a strong chondrogenic response leading to terminal maturation, whereas recombinant Wnt5a induced a mild chondrogenic response without promoting hypertrophy. Moreover, Wnt5a suppressed BMP-2-mediated chondrocyte maturation, preventing the formation of fibrocartilaginous tissue in high-density cultures treated sequentially with BMP-2 and Wnt5a. Implantation of scaffoldless pellets of hESC-derived chondroprogenitors pretreated with BMP-2 followed by Wnt5a into rat chondral defects induced an articular-like phenotype in vivo. Together, the data establish a novel role for Wnt5a in controlling the progression from multipotency into an articular-like cartilage phenotype in vitro and in vivo. Stem Cells Translational Medicine 2017;6:40-50.

Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity.

The bHLH transcription factor Twist1 has emerged as a negative regulator of chondrogenesis in skeletal progenitor cells and as an inhibitor of maturation in growth plate chondrocytes. However, its role in articular cartilage remains obscure. Here we examine Twist1 expression during re-differentiation of expanded human articular chondrocytes, the distribution of Twist1 proteins in normal versus OA human articular cartilage, and its role in modulating OA development in mice. High levels of Twist1 transcripts were detected by qPCR analyses of expanded de-differentiated human articular chondrocytes that had acquired mesenchymal-like features. The induction of hallmark cartilage genes by Bmp-2 mediated chondrogenic differentiation was paralleled by the dramatic suppression of Twist1 in vitro. In normal human articular cartilage, Twist1-expressing chondrocytes were most abundant in the superficial zone with little to no expression in the middle and deep zones. However, our analyses revealed a higher proportion of deep zone articular chondrocytes expressing Twist1 in human OA cartilage as compared to normal articular cartilage. Moreover, Twist1 expression was prominent within proliferative cell clusters near fissure sites in more severely affected OA samples. To assess the role of Twist1 in OA pathophysiology, we subjected wild type mice and transgenic mice with gain of Twist1 function in cartilage to surgical destabilization of the medial meniscus. At 12 weeks post-surgery, micro-CT and histological analyses revealed attenuation of the OA phenotype in Twist1 transgenic mice compared to wild type mice. Collectively, the data reveal a role for Twist in articular cartilage maintenance and the attenuation of cartilage degeneration.

Expression profiling of substantia nigra in Parkinson disease, progressive supranuclear palsy, and frontotemporal dementia with parkinsonism.

BACKGROUND: Parkinson disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra. Genes contributing to rare mendelian forms of PD have been identified, but the genes involved in the more common idiopathic PD are not well understood. OBJECTIVES: To identify genes important to PD pathogenesis using microarrays and to investigate their potential to aid in diagnosing parkinsonism. DESIGN: Microarray expression analysis of postmortem substantia nigra tissue. PATIENTS: Substantia nigra samples from 14 unrelated individuals were analyzed, including 6 with PD, 2 with progressive supranuclear palsy, 1 with frontotemporal dementia with parkinsonism, and 5 control subjects. MAIN OUTCOME MEASURES: Identification of genes significantly differentially expressed (P<.05) using Affymetrix U133A microarrays. RESULTS: There were 142 genes that were significantly differentially expressed between PD cases and controls and 96 genes that were significantly differentially expressed between the combined progressive supranuclear palsy and frontotemporal dementia with parkinsonism cases and controls. The 12 genes common to all 3 disorders may be related to secondary effects. Hierarchical cluster analysis after exclusion of these 12 genes differentiated 4 of the 6 PD cases from progressive supranuclear palsy and frontotemporal dementia with parkinsonism. CONCLUSIONS: Four main molecular pathways are altered in PD substantia nigra: chaperones, ubiquitination, vesicle trafficking, and nuclear-encoded mitochondrial genes. These results correlate well with expression analyses performed in several PD animal models. Expression analyses have promising potential to aid in postmortem diagnostic evaluation of parkinsonism.