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

Two sides of a story: Mothers' and adolescents' agreement on child disclosure in immigrant and native families

Research on immigrant families often has suggested that the process of immigration can lead to a distancing of adolescents and their parents. This study examined the actual agreement of immigrant and native mother–adolescent dyads in their reports on children’s disclosure as an indicator for a trusting mother–child relationship. The research questions related to group-level differences (immigrant vs. native dyads) in mother–adolescent agreement, the prediction of interdyadic differences in mother–adolescent agreement, and the associations between mother–adolescent agreement and both family conflicts and adolescents’ depressive symptoms. The sample was comprised of mother–adolescent dyads: 197 native German dyads (adolescents: mean age 14.7 years, 53 % female) and 185 immigrant dyads from the former Soviet Union (adolescents: mean age 15.7 years, 60 % female). Agreement was assessed using the intraclass correlation coefficient. The results revealed that mother–adolescent agreement was lower in immigrant dyads than in native dyads. In both samples, higher levels of adolescent autonomy predicted lower mother–adolescent agreement. Among immigrants, language brokering was an additional predictor of lower levels of mother–adolescent agreement. The interaction of language brokering and autonomy also turned out to be significant, indicating that if an adolescent was high in language brokering or autonomy, the effect of the other variable was negligible. In both groups, mother–adolescent agreement was negatively related to family conflicts. The study shows that processes in immigrant and native families are rather similar, but that in immigrant families some additional acculturation-related factors have to be considered for a full understanding of family dynamics

Immune Players in the CNS:The Astrocyte

<p>In the finely balanced environment of the central nervous system astrocytes, the most numerous cell type, play a role in regulating almost every physiological system. First found to regulate extracellular ions and pH, they have since been shown to regulate neurotransmitter levels, cerebral blood flow and energy metabolism. There is also growing evidence for an essential role of astrocytes in central immunity, which is the topic of this review. In the healthy state, the central nervous system is potently anti-inflammatory but under threat astrocytes readily respond to pathogens and to both sterile and pathogen-induced cell damage. In response, astrocytes take on some of the roles of immune cells, releasing cyto- and chemokines to influence effector cells, modulating the blood-brain barrier and forming glial scars. To date, much of the data supporting a role for astrocytes in immunity have been obtained from in vitro systems; however data from experimental models and clinical samples support the suggestion that astrocytes perform similar roles in more complex environments. This review will discuss some aspects of the role of astrocytes in central nervous system immunity.</p>