Nico Scherf

In vivo time-lapse imaging shows diverse niche engagement by quiescent and naturally activated hematopoietic stem cells

Hematopoietic stem cells (HSCs) maintain the turnover of mature blood cells during steady state and in response to systemic perturbations such as infections. Their function critically depends on complex signal exchanges with the bone marrow (BM) microenvironment in which they reside, but the cellular mechanisms involved in HSC-niche interactions and regulating HSC function in vivo remain elusive. We used a natural mouse parasite, Trichinella spiralis, and multipoint intravital time-lapse confocal microscopy of mouse calvarium BM to test whether HSC-niche interactions may change when hematopoiesis is perturbed. We find that steady-state HSCs stably engage confined niches in the BM whereas HSCs harvested during acute infection are motile and therefore interact with larger niches. These changes are accompanied by increased long-term repopulation ability and expression of CD44 and CXCR4. Administration of a CXCR4 antagonist affects the duration of HSC-niche interactions. These findings suggest that HSC-niche interactions may be modulated during infection.

Imaging, quantification and visualization of spatio-temporal patterning in mESC colonies under different culture conditions

Motivation: Mouse embryonic stem cells (mESCs) have developed into a prime system to study the regulation of pluripotency in stable cell lines. It is well recognized that different, established protocols for the maintenance of mESC pluripotency support morphologically and functionally different cell cultures. However, it is unclear how characteristic properties of cell colonies develop over time and how they are re-established after cell passage depending on the culture conditions. Furthermore, it appears that cell colonies have an internal structure with respect to cell size, marker expression or biomechanical properties, which is not sufficiently understood. The analysis of these phenotypic properties is essential for a comprehensive understanding of mESC development and ultimately requires a bioinformatics approach to guarantee reproducibility and high-throughput data analysis. Results: We developed an automated image analysis and colony tracking framework to obtain an objective and reproducible quantification of structural properties of cell colonies as they evolve in space and time. In particular, we established a method that quantifies changes in colony shape and (internal) motion using fluid image registration and image segmentation. The methodology also allows to robustly track motion, splitting and merging of colonies over a sequence of images. Our results provide a first quantitative assessment of temporal mESC colony formation and estimates of structural differences between colony growth under different culture conditions. Furthermore, we provide a stream-based visualization of structural features of individual colonies over time for the whole experiment, facilitating visual comprehension of differences between experimental conditions. Thus, the presented method establishes the basis for the model-based analysis of mESC colony development. It can be easily extended to integrate further functional information using fluorescence signals and differentiation markers. Availability: The analysis tool is implemented C++ and Mathematica 8.0 (Wolfram Research Inc., Champaign, IL, USA). The tool is freely available from the authors. We will also provide the source code upon request. Contact: nico.scherf@tu-dresden.de

Nimodipine enhances neurite outgrowth in dopaminergic brain slice co-cultures.

Calcium ions (Ca2+) play important roles in neuroplasticity and the regeneration of nerves. Intracellular Ca2+ concentrations are regulated by Ca2+ channels, among them L‐type voltage‐gated Ca2+ channels, which are inhibited by dihydropyridines like nimodipine. The purpose of this study was to investigate the effect of nimodipine on neurite growth during development and regeneration. As an appropriate model to study neurite growth, we chose organotypic brain slice co‐cultures of the mesocortical dopaminergic projection system, consisting of the ventral tegmental area/substantia nigra and the prefrontal cortex from neonatal rat brains. Quantification of the density of the newly built neurites in the border region (region between the two cultivated slices) of the co‐cultures revealed a growth promoting effect of nimodipine at concentrations of 0.1 μM and 1 μM that was even more pronounced than the effect of the growth factor NGF.

Phosphodiesterase 2 inhibitors promote axonal outgrowth in organotypic slice co-cultures.

The development of appropriate models assessing the potential of substances for regeneration of neuronal circuits is of great importance. Here, we present procedures to analyze effects of substances on fiber outgrowth based on organotypic slice co-cultures of the nigrostriatal dopaminergic system in combination with biocytin tracing and tyrosine hydroxylase labeling and subsequent automated image quantification. Selected phosphodiesterase inhibitors (PDE-Is) were studied to identify their potential growth-promoting capacities. Immunohistochemical methods were used to visualize developing fibers in the border region between ventral tegmental area/substantia nigra co-cultivated with the striatum as well as the cellular expression of PDE2A and PDE10. The quantification shows a significant increase of fiber density in the border region induced by PDE2-Is (BAY60-7550; ND7001), comparable with the potential of the nerve growth factor and in contrast to PDE10-I (MP-10). Analysis of tyrosine hydroxylase-positive fibers indicated a significant increase after treatment with BAY60-7550 and nerve growth factor in relation to dimethyl sulfoxide. Additionally, a dose-dependent increase of intracellular cGMP levels in response to the applied PDE2-Is in PDE2-transfected HEK293 cells was found. In summary, our findings show that PDE2-Is are able to significantly promote axonal outgrowth in organotypic slice co-cultures, which are a suitable model to assess growth-related effects in neuro(re)generation.

P2Y1 receptor mediated neuronal fibre outgrowth in organotypic brain slice co-cultures

Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y1R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y1R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPβS (P2Y1,12,13R agonist). The observed stimulatory potential of ADPβS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y1R knockout (P2Y1R(-/-)) mice, the ADPβS-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y1R. Using wortmannin and PD98059 we further showed that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPβS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y1R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration.

Assisting the Machine Paradigms for Human-Machine Interaction in Single Cell Tracking

Single cell tracking emerged as one of the fundamental experimental techniques over the past years in basic life science research. Though a large number of automated tracking methods has been introduced, they are still lacking the accuracy to reliably track complete cellular genealogies over many generations. Manual tracking on the other hand is tedious and slow. Semi-automated approaches to cell tracking are a good compromise to obtain comprehensive information in feasible amounts of time. In this work, we investigate the efficacy of different interaction paradigms for manual correction and processing of precomputed tracking results and present a respective tool that implements those strategies.

Single cell phenotyping reveals heterogeneity among haematopoietic stem cells following infection.

The hematopoietic stem cell (HSC) niche provides essential microenvironmental cues for the production and maintenance of HSCs within the bone marrow. During inflammation, hematopoietic dynamics are perturbed, but it is not known whether changes to the HSC–niche interaction occur as a result. We visualize HSCs directly in vivo, enabling detailed analysis of the 3D niche dynamics and migration patterns in murine bone marrow following Trichinella spiralis infection. Spatial statistical analysis of these HSC trajectories reveals two distinct modes of HSC behavior: (a) a pattern of revisiting previously explored space and (b) a pattern of exploring new space. Whereas HSCs from control donors predominantly follow pattern (a), those from infected mice adopt both strategies. Using detailed computational analyses of cell migration tracks and life‐history theory, we show that the increased motility of HSCs following infection can, perhaps counterintuitively, enable mice to cope better in deteriorating HSC–niche microenvironments following infection. Stem Cells 2017;35:2292–2304

Beyond genealogies: Mutual information of causal paths to analyse single cell tracking data

Single cell tracking, based on the computerised analysis of time-lapse movies, is a sophisticated experimental technique to quantify single cell dynamics in time and space. Although the resulting cellular genealogies comprehensively describe the divisional history of each cell, there are many open questions regarding the statistical analysis of this type of data. In particular, it is unclear, how tracking uncertainties or spatial information of cellular development can correctly be incorporated into the analysis. Here we propose a generalised description of single cell tracking data by spatiotemporal networks that can account for ambiguities in cell assignment as well as for spatial relations between cells. We present a way to measure correlations among cell states by analysing the mutual information in state space considering causal (time-respecting) paths and illustrate our approach by a corresponding example. We conclude that a comprehensive spatiotemporal description of single cell tracking data is ultimately necessary to fully exploit the information obtained by time-lapse imaging.

Segmentation of Axonal Fibres in Tissue Slices

This work focuses on the segmentation of axonal structures in digital images of organotypic slice co-cultures. An image processing chain is presented, which relies on anisotropic diffusion for preprocessing of the images and the intelligent scissors method for segmentation. This method requires manual user interaction to set the starting points. To overcome this drawback, the initial parameters for the intelligent scissors are automatically extracted from the images by a graph-based approach.

Biology-inspired visualization of morphogenetic motion in the zebrafish endoderm

Effective visualization of large, multidimensional image data is a key instrument for data exploration in biological and medical applications. We propose a method to visualize motion patterns on a spherical surface, illustrated for the example of the zebrafish embryo. In particular, we analyze cellular motion by integrating prior knowledge about the underlying biological process and show how this improves visualization and connects to the conceptional description of the gastrulation process. We conclude that specialized visualizations are necessary to match data representation with conceptional ideas and to get a more intuitive understanding of the design principles of embryonic tissue formation.