Kristof Notelaers

Complex invasion pattern of the cerebral cortex bymicroglial cells during development of the mouse embryo.

Microglia are the immune cells of the central nervous system. They are suspected to play important roles in adult synaptogenesis and in the development of the neuronal network. Microglial cells originate from progenitors in the yolk sac. Although it was suggested that they invade the cortex at early developmental stages in the embryo, their invasion pattern remains largely unknown. To address this issue we analyzed the pattern of cortical invasion by microglial cells in mouse embryos at the onset of neuronal cell migration using in vivo immunohistochemistry and ex vivo time‐lapse analysis of microglial cells. Microglial cells begin to invade the cortex at 11.5 days of embryonic age (E11.5). They first accumulate at the pial surface and within the lateral ventricles, after which they spread throughout the cortical wall, avoiding the cortical plate region in later embryonic ages. The invasion of the cortical parenchyma occurs in different phases. First, there is a gradual increase of microglial cells between E10.5 and E14.5. From E14.5 to E15.5 there is a rapid phase with a massive increase in microglia, followed by a slow phase again from E15.5 until E17.5. At early stages, many peripheral microglia are actively proliferating before entering the parenchyma. Remarkably, activated microglia accumulate in the choroid plexus primordium, where they are in the proximity of dying cells. Time‐lapse analysis shows that embryonic microglia are highly dynamic cells.

Cardiac atrial appendage stem cells engraft and differentiate into cardiomyocytes in vivo: A new tool for cardiac repair after MI.

BACKGROUND This study assessed whether autologous transplantation of cardiac atrial appendage stem cells (CASCs) preserves cardiac function after myocardial infarction (MI) in a minipig model. METHODS AND RESULTS CASCs were isolated from right atrial appendages of Göttingen minipigs based on high aldehyde dehydrogenase activity and expanded. MI was induced by a 2h snare ligation of the left anterior descending coronary artery. Upon reperfusion, CASCs were intramyocardially injected under NOGA guidance (MI-CASC, n=10). Non-transplanted pigs (MI, n=8) received sham treatment. 3D electromechanical mapping (EMM) and cardiac MRI were performed to assess left ventricular (LV) function. MI pigs developed LV dilatation at 2 months (2M), while in the MI-CASC group volumes remained stable. Global LV ejection fraction decreased by 16 ± 8% in MI animals vs 3 ± 10% in MI-CASC animals and regional wall thickening in border areas was better preserved in the MI-CASC group. EMM showed decreased viability and wall motion in the LV for both groups POST-MI, whereas at 2M these parameters only improved in the MI-CASC. Substantial cell retention was accompanied by cardiomyogenic differentiation in 98±1% of the transplanted CASCs, which functionally integrated. Second harmonic generation microscopy confirmed the formation of mature sarcomeres in transplanted CASCs. Absence of cardiac arrhythmias indicated the safety of CASC transplantation. CONCLUSION CASCs preserve cardiac function by extensive engraftment and cardiomyogenic differentiation. Our data indicate the enormous potential of CASCs in myocardial repair.

Intracellular dynamics and fate of polystyrene nanoparticles in A549 Lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking.

Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems.

Possibilities and limitations for co-transplantation of cardiac atrial appendage stem cells and mesenchymal stem cells for myocardial repair

[Fanton, Yanick; Rummens, Jean-Luc; Daniels, Annick; Windmolders, Severina; Willems, Leen; Declercq, Jeroen; Hensen, Karen; Koninckx, Remco] Jessa Hosp, Lab Expt Hematol, Hasselt, Belgium. [Fanton, Yanick; Robic, Boris; Rummens, Jean-Luc; Windmolders, Severina; Willems, Leen; Notelaers, Kristof; Paesen, Rik; Ameloot, Marcel; Bito, Virginie; Declercq, Jeroen; Hensen, Karen; Koninckx, Remco; Hendrikx, Marc] Hasselt Univ, Fac Med & Life Sci, Hasselt, Belgium. [Robic, Boris; Mees, Urbain; Hendrikx, Marc] Jessa Hosp, Dept Cardiothorac Surg, Hasselt, Belgium. [Jamaer, Luc; Dubois, Jasperina] Jessa Hosp, Dept Cardiac Anesthesia, Hasselt, Belgium. [Bijnens, Eric; Heuts, Nic] Jessa Hosp, Dept Radiol, MRI Unit, Hasselt, Belgium. [Notelaers, Kristof; Paesen, Rik; Ameloot, Marcel; Bito, Virginie] Hasselt Univ, Biomed Res Inst, Hasselt, Belgium.

On the interpretation of second harmonic generation intensity profiles of striated muscle

Abstract. Recently, a supramolecular model was developed for predicting striated skeletal muscle intensity profiles obtained by label-free second harmonic generation (SHG) microscopy. This model allows for a quantitative determination of the length of the thick filament antiparallel range or M-band (M), and results in M=0.12  μm for single-band intensity profiles when fixing the A-band length (A) to A=1.6  μm, a value originating from electron microscopy (EM) observations. Using simulations and experimental data sets, we showed that the objective numerical aperture (NA) and the refractive index (RI) mismatch (Δn=n2ω−nω) between the illumination wave (ω) and the second harmonic wave (2ω) severely affect the simulated sarcomere intensity profiles. Therefore, our recovered filament lengths did not match with those observed by EM. For an RI mismatch of Δn=0.02 and a moderate illumination NA of 0.8, analysis of single-band SHG intensity profiles with freely adjustable A- and M-band sizes yielded A=1.40±0.04  μm and M=0.07±0.05  μm for skeletal muscle. These lower than expected values were rationalized in terms of the myosin density distribution along the myosin thick filament axis. Our data provided new and practical insights for the application of the supramolecular model to study SHG intensity profiles in striated muscle.

Automatic particle detection in microscopy using temporal correlations

One of the fundamental problems in the analysis of single particle tracking data is the detection of individual particle positions from microscopy images. Distinguishing true particles from noise with a minimum of false positives and false negatives is an important step that will have substantial impact on all further analysis of the data. A common approach is to obtain a plausible set of particles from a larger set of candidate particles by filtering using manually selected threshold values for intensity, size, shape, and other parameters describing a particle. This introduces subjectivity into the analysis and hinders reproducibility. In this paper, we introduce a method for automatic selection of these threshold values based on maximizing temporal correlations in particle count time series. We use Markov Chain Monte Carlo to find the threshold values corresponding to the maximum correlation, and we study several experimental data sets to assess the performance of the method in practice by comparing manually selected threshold values from several independent experts with automatically selected threshold values. We conclude that the method produces useful results, reducing subjectivity and the need for manual intervention, a great benefit being its easy integratability into many already existing particle detection algorithms. Microsc. Res. Tech., 76:997–1006, 2013.