Isolation, selection and culture methods to enhance clonogenicity of mouse bone marrow derived mesenchymal stromal cell precursors.

INTRODUCTION: Conventionally cultured mouse bone marrow mesenchymal stromal cells (mBM-MSC) are a heterogeneous population that often initially contain contaminating haematopoietic cells. Variability in isolation methods, culture protocols and the lack of specific mBM MSC markers might explain this heterogeneity. The aim of this study is to optimise the isolation, culture conditions and selection of mBM-MSC. METHODS: Mouse BM-MSCs were isolated from crushed long bones (cBM) or flushed bone marrow (fBM) from 6-8 week old C57Bl/6 mice. These subpopulations were analysed by flow cytometry using commonly used mBM-MSC cell surface marker, e.g. Sca-1, CD29 and CD44. Cells were cultured and expanded in vitro in hypoxic conditions of either 2 % or 5 % oxygen. Cell sorting and qRT-PCR was used to determine transcript levels of stem cell and lineage related genes in individual subpopulations. RESULTS: During early passaging not only do contaminating haematopoietic cells disappear, but there is a change in the phenotype of mBM-MSC affecting particularly CD44 and Sca-1 expression. By fluorescence activated cell sorting of CD45(-)/Ter119(-) mBM stroma based on Sca-1 expression and expansion in hypoxic conditions, we show that Sca-1(+) cells had higher CFU-F frequencies and showed enhanced proliferation compared with Sca-1(-) cells. As evaluated by in vitro assays and qRT-PCR, these cells presented in vitro tri-lineage differentiation along osteocyte, chondrocyte, and adipocyte lineages. Finally, by prospective isolation of Sca-1(+)PDGFRα(+)CD90(+) cells we have isolated mBM-MSC on a single cell level, achieving a CFU-F frequency of 1/4. Functional investigations demonstrated that these MSC clones inhibited T-lymphocyte proliferation. CONCLUSION: By positive selection using a combination of antibodies to Sca-1, CD90 and PDGFRα and culturing in hypoxia, we have found a subpopulation of BM cells from C57Bl/6 mice with a CFU-F cloning efficiency of 1/4. To our knowledge these results represent the highest frequencies of mouse MSC cloning from C57Bl/6 mice yet reported.

Effects of growth phase and temperature on σB activity within a Listeria monocytogenes population: evidence for RsbV-independent activation of σB at refrigeration temperatures.

The alternative sigma factor σB of Listeria monocytogenes is responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation of σB within populations of L. monocytogenes EGD-e wild-type, ΔsigB, and ΔrsbV throughout growth at both 4°C and 37°C, using a reporter fusion that couples expression of EGFP to the strongly σB-dependent promoter of lmo2230. A similar σB activation pattern within the population was observed in wt-egfp at both temperatures, with the highest induction of σB occurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction of σB activity was heterogeneous, with only a proportion of the cells in the wt-egfp population being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation of σB was observed during growth at 4°C. This result suggests that an alternative route to σB activation exists in the absence of RsbV, a finding that is not explained by the current model for σB regulation.

The influence of hypoxia on the differentiation capacities and immunosuppressive properties of clonal mouse mesenchymal stromal cell lines.

Multipotent mesenchymal stromal cells are multipotent cells capable of differentiating into different mesodermal cell types. Enigmatically, mesenchymal stromal cells present in the bone marrow support early lymphopoiesis yet can inhibit mature lymphocyte growth. Critical features of the bone marrow microenvironment, such as the level of oxygen, play an important role in mesenchymal stromal cell biology. Herein, we show that a panel of continuously growing mouse mesenchymal stromal cell lines, namely OP9, MS5, PA6, ST2 and B16-14, exhibit mesenchymal stromal cell characteristic phenotypes and respond physiologically to oxygen deprivation. Culturing freshly isolated bone marrow-derived mesenchymal stromal cells or cell lines at 5% O2 resulted in a dramatic increase in expression of hypoxia-inducible factor family members and of key genes involved in their differentiation. Phenotypically, their osteogenic and adipogenic differentiation capacity was generally improved in hypoxia, whereas their inhibitory effects on in vitro T-cell proliferation were preserved. Taken together, we conclude that these continuously growing mouse cell lines behave as canonical mesenchymal stromal cells and respond physiologically to hypoxia, thereby providing a potent tool for the study of different aspects of mesenchymal stromal cell biology.

Development and optimization of an EGFP-based reporter for measuring the general stress response in Listeria monocytogenes.

A characteristic of the food-borne pathogen Listeria monocytogenes is its tolerance to the harsh conditions found both in minimally processed foods and the human gastrointestinal tract. This trait is partly under the control of the alternative sigma factor sigma B (σ(B)). To study the mechanisms that trigger the activation of σ(B) , and hence the development of stress tolerance, we have developed a fluorescent reporter fusion that allows the real-time activity of σ(B) to be monitored. The reporter, designated Plmo2230::egfp, fuses the strong σ(B)-dependent promoter from the lmo2230 gene (which encodes a putative arsenate reductase) to a gene encoding enhanced green fluorescence protein (EGFP). The reporter was integrated into the genomes of the wild-type strain L. monocytogenes EGD-e as well as two mutant derivatives lacking either sigB or rsbV. The resulting strains were used to study σ(B) activation in response to growth phase and hyperosmotic stress. The wild-type was strongly fluorescent in stationary phase or in cultures with added NaCl and this fluorescence was abolished in both the sigB and rsbV backgrounds, consistent with the σ(B)-dependency of the lmo2230 promoter. During sudden osmotic upshock (addition of 0.5 M NaCl during growth) a real-time increase in fluorescence was observed microscopically, reaching maximal activation after 30 min. Flow cytometry was used to study the activation of σ(B) at a population level by hyperosmotic stress during exponential growth. A strong and proportional increase in fluorescence was observed as the salt concentration increased from 0 to 0.9 M NaCl. Interestingly, there was considerable heterogeneity within the population and a significant proportion of cells failed to induce a high level of fluorescence, suggesting that σ(B) activation occurs stochastically in response to hyperosmotic stress. Thus the Plmo2230::egfp is a powerful tool that will allow the stress response to be better studied in this important human pathogen.

Albumin dialysis molecular adsorbents recirculating system: impact of dialysate albumin concentration on detoxification efficacy.

Albumin dialysis with the molecular adsorbent recirculating system (MARS) or single pass albumin dialysis (SPAD) uses human serum albumin (HSA) as an addendum of the dialysate fluid. The purpose of this in vitro study was to evaluate the impact of the dialysate albumin concentration on removal efficacy. Heparinized human plasma (3 L/test) was spiked with creatinine (1000 mg/L), unconjugated bilirubin (100 mg/L), chenodeoxycholic acid (CDCA) (100 mg/L), and diazepam (3 mg/L). The MARS albumin circuit was primed with different amounts of HSA (150, 100, 60, and 40 g). The plasma, albumin, and dialysate flow rates were 200, 200, and 40 mL/min, respectively. Clearances were calculated based on repeated sampling during the experiments, which lasted 480 min. The effective HSA concentrations in the dialysate were 175, 115, 77, and 46 g/L, respectively. They decreased over treatment time to 147, 99, 63, and 41 g/L, respectively, due to surface adsorption. The plasma-HSA concentration remained unchanged over time in all experiments (average 39 g/L). The creatinine clearance was not impacted by dialysate HSA concentration. For the albumin-bound markers a clear correlation between HSA-concentration and clearance was demonstrated with the highest clearances for the 100 and 150 g HSA experiments. The 100 g HSA setup appeared to be the one with best cost-benefit ratio, resulting in clearances (after 1 h of treatment) of 31 mL/min creatinine, 0.3 mL/min unconjugated bilirubin, 11 mL/min CDCA, and 35 mL/min diazepam. Low albumin concentrations, such as in SPAD, result in low clearance rates for albumin-bound substances. The optimal clearances in these experiments were reached with a priming dose of 100 g HSA.