visualization-quantification-mesenchymal-cell-adipogenic

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

Several dyes are currently available for use in detecting differentiation of mesenchymal cells into adipocytes. Dyes, such as Oil Red O, are cheap, easy ...

The University of Auckland

The human brain is a highly vascular organ in which the blood-brain barrier (BBB) tightly regulates molecules entering the brain. Pericytes are an integral cell type of the BBB, regulating vascular integrity, neuroinflammation, angiogenesis and wound repair. Despite their importance, identifying pericytes amongst other perivascular cell types and deciphering their specific role in the neurovasculature remains a challenge. Using primary adult human brain cultures and fluorescent-activated cell sorting, we identified two CD73(+)CD45(-) mesenchymal populations that showed either high or low CD90 expression. CD90 is known to be present on neurons in the brain and peripheral blood vessels. We found in the human brain, that CD90 immunostaining localised to the neurovasculature and often associated with pericytes. In vitro, CD90(+) cells exhibited higher basal proliferation, lower expression of markers αSMA and CD140b, produced less extracellular matrix (ECM) proteins, and exhibited lesser pro-inflammatory responses when compared to the CD90(-) population. Thus, CD90 distinguishes two interrelated, yet functionally distinct pericyte populations in the adult human brain that may have discrete roles in neurovascular function, immune response and scar formation.

Cultured pericytes from human brain show phenotypic and functional differences associated with differential CD90 expression.

The combination of solid phase peptide synthesis and-β--acetylglucosaminidase (ENGase) catalysed glycosylation is a powerful convergent synthetic method allowing access to glycopeptides bearing full-length-glycan structures. Mannose-terminated-glycan oligosaccharides, produced by either total or semi-synthesis, were converted into oxazoline donor substrates. A peptide from the human cytomegalovirus (CMV) tegument protein pp65 that incorporates a well-characterised T cell epitope, containing-acetylglucosamine at specific Asn residues, was accessed by solid phase peptide synthesis, and used as an acceptor substrate. High-yielding enzymatic glycosylation afforded glycopeptides bearing defined homogeneous high-mannose-glycan structures. These high-mannose containing glycopeptides were tested for enhanced targeting to human antigen presenting cells (APCs), putatively mediatedthe mannose receptor, and for processing by the APCs for presentation to human CD8+ T cells specific for a 9-mer epitope within the peptide. Binding assays showed increased binding of glycopeptides to APCs compared to the non-glycosylated control. Glycopeptides bearing high-mannose-glycan structures at a single site outside the T cell epitope were processed and presented by the APCs to allow activation of a T cell clone. However, the addition of a second glycan within the T cell epitope resulted in ablation of T cell activation. We conclude that chemo-enzymatic synthesis of mannosylated glycopeptides enhances uptake by human APCs while preserving the immunogenicity of peptide epitopes within the glycopeptides, provided those epitopes are not themselves glycosylated.

Convergent chemo-enzymatic synthesis of mannosylated glycopeptides; targeting of putative vaccine candidates to antigen presenting cells.

Lymphatic drainage from skin on the head and neck is complex. We sought to provide improved visualization and analysis of the patterns of head and neck skin lymphatic drainage using aggregated lymphoscintigraphy data.

Three-dimensional visualization of skin lymphatic drainage patterns of the head and neck.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) acts through tumor vascular disruption and cytokine production and is the first of its class to enter phase 3 trials. We characterized leukocytes and cytokines in murine Colon 38 tumors before and after DMXAA treatment. Tumor mass declined 50% 24 hours after DMXAA administration, but the leukocyte count per gram of tumor increased threefold owing to a large influx of Ly6G(+)CD11b(+)F4/80(-) cells with the morphology of neutrophils. However, B and T lymphocytes, natural killer cells, and macrophages in the tumor all decreased in numbers. Seven chemokines were substantially induced in the tumor, spleen, and serum 4 hours after DMXAA administration. Using cultured spleen cell subpopulations, CD11b(+) cells (largely monocytes and macrophages) were shown to be the primary producers of tumor necrosis factor alpha, interleukin 6 (IL-6), and macrophage inflammatory 1alpha (MIP-1alpha). CD49b(+) natural killer cells produced IP-10, whereas CD45R(+) B lymphocytes produced regulated upon activation normal T cell express sequence. T lymphocytes were not major producers of cytokines in the response to DMXAA. Murine peripheral blood leukocytes (PBLs) produced a similar panel of cytokines in culture to that detected in mouse serum after DMXAA treatment. Cytokines in human PBL cultures were subsequently measured with the aim of identifying potential serum markers of the human response to DMXAA. IP-10 (P < .001), monocyte chemoattractant protein 1 (P < .001), and sCD40L (P < .01) were decreased, whereas IL-8 (P < .001) and MIP-1alpha (P = .03) were increased in DMXAA-treated compared with untreated PBL cultures from a group of 12 donors.

Neutrophil influx and chemokine production during the early phases of the antitumor response to the vascular disrupting agent DMXAA (ASA404).

A radical lipidation: Application of a novel thiol-ene lipidation enables the one-step synthesis of self-adjuvanting antigenic peptides as vaccine candidates. The resultant monoacyl lipopeptides are shown to activate monocytes in a robust manner.

Direct peptide lipidation through thiol-ene coupling enables rapid synthesis and evaluation of self-adjuvanting vaccine candidates.

We have detected intramucosal bacteria within the sinus mucosa of patients with chronic rhinosinusitis (CRS), but our attempts at characterizing these did not yield any discernible genotypic or phenotypic differences from surface bacteria. We hypothesized that the presence of intramucosal microcolonies reflected host mucosal immune dysfunction. This study characterizes the activation status of T cells, B cells, and macrophages in the sinus mucosa of patients with CRS and controls and determines the impact of bacteria on mucosal immunology.

The interaction between bacteria and mucosal immunity in chronic rhinosinusitis: a prospective cross-sectional analysis.

The lymphatic sinuses in human lymph nodes (LNs) are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs). A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs); in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs) in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.

Mapping the distinctive populations of lymphatic endothelial cells in different zones of human lymph nodes.

microRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation. We also investigated how changes in miR-146a related to other miRNAs that alter with CD8+ differentiation status.

MicroRNA regulation in human CD8+ T cell subsets--cytokine exposure alone drives miR-146a expression.

Tearing of the rotator cuff tendon in the shoulder is a significant clinical problem, with large/full-thickness tears present in ∼22% of the general population and recurrent tear rates postarthroscopic repair being quoted as high as 94%. Tissue-engineered biomaterials are increasingly being investigated as a means to augment rotator cuff repairs, with the aim of inducing host cell responses to increase tendon tissue regeneration. Silk-derived materials are of particular interest due to the high availability, mechanical strength, and biocompatibility of silks. In this study, Spidrex(®), a novel knitted, non-mulberry silk fibroin scaffold was evaluated in vitro for its potential to improve tendon regeneration. Spidrex was compared with a knitted Bombyx mori silk scaffold, a 3D collagen gel and Fiberwire(®) suture material. Primary human and rat tenocytes successfully adhered to Spidrex and significantly increased in number over a 14 day period (p<0.05), as demonstrated by fluorescent calcein-AM staining and alamarBlue(®) assays. A similar growth pattern was observed with human tenocytes cultured on the B. mori scaffold. Morphologically, human tenocytes elongated along the silk fibers of Spidrex, assuming a tenocytic cell shape, and were less circular with a higher aspect ratio compared with human tenocytes cultured on the B. mori silk scaffold and within the collagen gel (p<0.05). Gene expression analysis by real-time PCR showed that rat tenocytes cultured on Spidrex had increased expression of tenocyte-related genes such as fibromodullin, scleraxis, and tenomodulin (p<0.05). Expression of genes that indicate transdifferentiation toward a chondrocytic or osteoblastic lineage were significantly lower in tenocytes cultured on Spidrex in comparison to the collagen gel (p<0.05). Immunogenicity assessment by the maturation of and cytokine release from primary human dendritic cells demonstrated that Spidrex enhanced dendritic cell maturation in a similar manner to the clinically used suture material Fiberwire, and significantly upregulated the release of proinflammatory cytokines (p<0.05). This suggests that Spidrex may induce an early immune response postimplantation. While further work is required to determine what effect this immune response has on the tendon healing process, our in vitro data suggests that Spidrex may have the cytocompatibility and bioactivity required to support tendon regeneration in vivo.

In vitro evaluation of a novel non-mulberry silk scaffold for use in tendon regeneration.

Herein, we report the facile preparation of cell-targeted platinum nanoparticles (PtNPs), through the design of peptides that, as a single molecule added in small concentration during the synthesis, control the size of PtNP clusters during their growth, stabilise the PtNPs in aqueous suspension and enable the functionalisation of the PtNPs with a versatile range of cell-targeting ligands. Water-soluble PtNPs targeted respectively at blood group antigens and at integrin receptors are demonstrated.

Cell-targeted platinum nanoparticles and nanoparticle clusters.

The skin is largely comprised of keratinocytes within the interfollicular epidermis. Over approximately two weeks these cells differentiate and traverse the thickness of the skin. The stage of differentiation is therefore reflected in the positions of cells within the tissue, providing a convenient axis along which to study the signaling events that occur in situ during keratinocyte terminal differentiation, over this extended two-week timescale. The canonical ERK-MAPK signaling cascade (Raf-1, MEK-1/2 and ERK-1/2) has been implicated in controlling diverse cellular behaviors, including proliferation and differentiation. While the molecular interactions involved in signal transduction through this cascade have been well characterized in cell culture experiments, our understanding of how this sequence of events unfolds to determine cell fate within a homeostatic tissue environment has not been fully characterized.

Regulation of ERK-MAPK signaling in human epidermis.

Rotator cuff tears can cause significant pain and functional impairment. Without surgical repair, the rotator cuff has little healing potential, and following surgical repair, they are highly prone to re-rupture. Augmenting such repairs with a biomaterial scaffold has been suggested as a potential solution. Extracellular matrix (ECM)-based scaffolds are the most commonly used rotator cuff augments, although to date, reports on their success are variable. Here, we utilize pre-clinical in vitro and in vivo assays to assess the efficacy of a novel biomaterial scaffold, ovine forestomach extracellular matrix (OFM), in augmenting rotator cuff repair.

P. Rod Dunbar

School of Biological Sciences,
Maurice Wilkins Centre

The University of Auckland

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

P. Rod Dunbar

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The University of Auckland

Visualization and Quantification of Mesenchymal Cell Adipogenic Differentiation Potential with a Lineage Specific Marker

School of Biological Sciences,
Maurice Wilkins Centre