Apoptosis, mastocytosis, and diminished adipocytokine gene expression accompany reduced epididymal fat mass in long-standing diet-induced obese mice

<p>Abstract</p> <p>Background</p> <p>Obesity is characterized by increased cell death and inflammatory reactions in the adipose tissue. Here, we explored pathophysiological alterations taking place in the adipose tissue in long-standing obesity. In the epididymal fat of C57BL/6 mice fed a high-fat diet for 20 weeks, the prevalence and distribution of dead adipocytes (crown-like structures), mast cells (toluidine blue, mMCP6), macrophages (F4/80), and apoptotic cells (cleaved caspase-3) were measured. Moreover, gene and/or protein expression of several adipocytokines (leptin, adiponectin, TNF-α, IL-10, IL-6, MCP-1), F4/80, mMCP6, cleaved caspase-3 were determined.</p> <p>Results</p> <p>We observed that the epididymal fat mass was lower in obese than in lean mice. In obese mice, the epididymal fat mass correlated inversely with body weight and liver mass. Dead adipocytes, mast cells, macrophages, and apoptotic cells were abundant in the epididymal fat of obese mice, especially in the rostral vs. caudal zone. Accordingly, mMCP6, F4/80, and cleaved caspase-3 gene and/or protein expression was increased. Conversely, adiponectin, leptin, IL-6, and MCP-1 gene expression levels were lower in the epididymal fat of obese than lean mice. Although TNF-α and IL-10 gene expression was higher in the epididymal fat of obese mice, their expression relative to F4/80 and mMCP6 expression were lower in the heavily infiltrated rostral than caudal zone.</p> <p>Conclusions</p> <p>This study demonstrates that in mice with long-standing obesity diminished gene expression of several adipocytokines accompany apoptosis and reduced mass of the epididymal fat. Our findings suggest that this is due to both increased prevalence of dead adipocytes and altered immune cell activity. Differential distribution of metabolically challenged adipocytes is indicative of the presence of biologically diverse zones within the epididymal fat.</p

Atlante delle residenze collettive

Atlante delle residenze colletiv

Teaching Real Data Interpretation with Models (TRIM): Analysis of Student Dialogue in a Large-Enrollment Cell and Developmental Biology Course

We present our design for a cell biology course to integrate content with scientific practices, specifically data interpretation and model-based reasoning. A 2-yr research project within this course allowed us to understand how students interpret authentic biological data in this setting. Through analysis of written work, we measured the extent to which students' data interpretations were valid and/or generative. By analyzing small-group audio recordings during in-class activities, we demonstrated how students used instructor-provided models to build and refine data interpretations. Often, students used models to broaden the scope of data interpretations, tying conclusions to a biological significance. Coding analysis revealed several strategies and challenges that were common among students in this collaborative setting. Spontaneous argumentation was present in 82% of transcripts, suggesting that data interpretation using models may be a way to elicit this important disciplinary practice. Argumentation dialogue included frequent co-construction of claims backed by evidence from data. Other common strategies included collaborative decoding of data representations and noticing data patterns before making interpretive claims. Focusing on irrelevant data patterns was the most common challenge. Our findings provide evidence to support the feasibility of supporting students' data-interpretation skills within a large lecture course.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Design of a stirred multiwell bioreactor for expansion of CD34(+) umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O(2) concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O(2) solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O(2) concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34(+) cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O(2) for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34(+) cells at 5% O(2) , using 21%-O(2) cultures as a control. In hypoxia we found that CD34(+) cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen- and c-kit-dependent pathways at the molecular level

Design of a stirred multiwell bioreactor for expansion of CD34(+) umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O(2) concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O(2) solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O(2) concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34(+) cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O(2) for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34(+) cells at 5% O(2) , using 21%-O(2) cultures as a control. In hypoxia we found that CD34(+) cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen- and c-kit-dependent pathways at the molecular level

Confined 3D microenvironment regulates early differentiation in human pluripotent stem cells

The therapeutic potential of human pluripotent stem (hPS) cells is threatened, among various problems, by the difficulty to homogenously direct cell differentiation into specific lineages. The transition from hPSC into committed differentiated cells is accompanied by secretome activity, remodeling of extracellular matrix and self-organization into germ layers. In this work, we aimed to investigate how different three-dimensional microenvironments regulate the early differentiation of the three germ layers in human embryonic stem (hES) cells derived embryoid bodies. In particular, a permeable, biocompatible, hydrogel microwell array was specifically designed for recreating a confined niche in which EB secreted molecules accumulate in accordance with hydrogel diffusional cut-off. Fluorescence recovery after photobleaching technique was performed to accurately evaluate hydrogel permeability, mesh size and diffusional cutoff for soluble molecules. Three different culture conditions of EB culture were analyzed: suspension, confinement in microwells of width/depth ratio 1:1 and 1:2. Results show that EBs cultured in microwells are viable and have comparable average size after 8 days culture. Whole genome microarrays show that significative differential gene expression was observed between suspension and confined EBs culture. In particular, EBs culture in microwells promotes the expression of genes involved in pattern specification processes, brain development, ectoderm and endoderm differentiation. On the contrary, suspension EBs express instead genes involved in mesoderm specification and heart development. These results suggest that local accumulation of EBs secreted molecules drives differentiation patterns, as confirmed by immunofluorescence of germ layer markers, in hydrogel confined EB culture from both hES cells and human induced pluripotent stem (hiPS) cells. Our findings highlight an additional potential role of biomaterial in controlling hPSC differentiation through secreted factor niche specification