Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

Variation in population levels of sedentary time in European children and adolescents according to cross-European studies: a systematic literature review within DEDIPAC

peer-reviewedBackground: A high amount of sedentary time has been proposed as a risk factor for various health outcomes in adults. While the evidence is less clear in children and adolescents, monitoring sedentary time is important to understand the prevalence rates and how this behaviour varies over time and by place. This systematic literature review aims to provide an overview of existing cross-European studies on sedentary time in children (0-12y) and adolescents (13-18y), to describe the variation in population levels of sedentary time, and to discuss the impact of assessment methods. Methods: Six literature databases were searched (PubMed, EMBASE, CINAHL, PsycINFO, SportDiscus and OpenGrey), followed by backward- and forward tracking and searching authors’ and experts’ literature databases. Included articles were observational studies reporting on levels of sedentary time in the general population of children and/or adolescents in at least two European countries. Population levels were reported separately for children and adolescents. Data were reviewed, extracted and assessed by two researchers, with disagreements being resolved by a third researcher. The review protocol is published under registration number CRD42014013379 in the PROSPERO database. Forty-two eligible articles were identified, most were cross-sectional (n = 38). The number of included European countries per article ranged from 2 to 36. Levels of sedentary time were observed to be higher in East-European countries compared to the rest of Europe. There was a large variation in assessment methods and reported outcome variables. The majority of articles used a child-specific questionnaire (60 %). Other methods included accelerometers, parental questionnaires or interviews and ecological momentary assessment tools. Television time was reported as outcome variable in 57 % of included articles (ranging from a mean value of 1 h to 2.7 h in children and 1.3 h to 4.4 h in adolescents), total sedentary time in 24 % (ranging from a mean value of 192 min to 552 min in children and from 268 min to 506 min in adolescents). A substantial number of published studies report on levels of sedentary time in children and adolescents across European countries, but there was a large variation in assessment methods. Questionnaires (child specific) were used most often, but they mostly measured specific screen-based activities and did not assess total sedentary time. There is a need for harmonisation and standardisation of objective and subjective methods to assess sedentary time in children and adolescents to enable comparison across countries

Histamine Derived from Probiotic Lactobacillus reuteri Suppresses TNF via Modulation of PKA and ERK Signaling

Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases