Heterogeneity in astrocyte responses after acute injury in vitro and in vivo

Astrocytes present a major population of glial cells in the adult mammalian brain. The heterogeneity of astrocytes in different regions of the healthy central nervous system (CNS) and their physiological functions are well understood. In contrast, rather little is known about the diversity of astrocyte reactions under pathological conditions. After CNS injury the reaction of astrocytes, also termed ‘reactive astrogliosis’, is characterized by morphological and molecular changes such as hypertrophy, polarization, migration and up-regulation of intermediate filaments. So far, it was unknown whether all astrocytes undergo these changes, or whether only specific subpopulations of reactive astrocytes possess special plasticity. Since some quiescent, postmitotic astrocytes in the cortical gray matter apparently de-differentiate and re-enter the cell cycle upon injury, reactive astrocytes have the ability to acquire restrictive stem cell potential. However, the mechanisms leading to increased astrocyte numbers after acute injury, e.g. proliferation and migration, had not been investigated live in vivo. For the first time, recently established in vivo imaging using 2-photon laser scanning microscopy (2pLSM) allowed to follow single GFP-labeled astrocytes for days and weeks after cortical stab wound injury. Tracing morphological changes during the transition from a quiescent to reactive state, these live observations revealed a heterogeneous behavior of reactive astrocytes depending on the lesion size. Different subsets of astrocytes either became hypertrophic, polarized and/ or divided, but never migrated towards the injury. Intriguingly, the lack of astrocyte migration was not only contradictory to what had been predicted based on in vitro and in situ studies, but was also in stark contrast to the motility of other glial cells. Additionally, live imaging provided first evidence that only a small subset of reactive astrocytes in juxtavascular positions re-gains proliferative capacity after injury. While astrocyte proliferation was affected by conditional deletion of RhoGTPase Cdc42 – a key regulator of cell polarity –, the vascular niche was preserved, indicating that juxtavascular astrocytes are uniquely suited for proliferation after injury. Following the behavior of cdc42-deficient astrocytes by live imaging using an in vitro scratch wound assay, cell-autonomous effects including disturbed polarity and impaired directional migration confirmed a crucial role of Cdc42 signaling in reactive astrocytes after acute injury in vitro and in vivo. These novel insights revise current concepts of reactive astrocytes involved in glial scar formation by assigning regenerative potential to a minor pool of proliferative, juxtavascular astrocytes, and suggesting specific functions of different astrocyte subsets after CNS trauma.Astrozyten bilden die größte Gruppe von Gliazellen im Gehirn erwachsener Säugetiere. Die Heterogenität von Astrozyten in verschiedenen Regionen des zentralen Nervensystems (ZNS), sowie deren physiologischen Funktionen sind relativ gut untersucht. Hingegen ist die Diversität der astrozytären Reaktion unter pathologischen Bedingungen bis jetzt wenig verstanden. In Folge einer Verletzung des ZNS reagieren Astrozyten mit bestimmten molekularen und morphologischen Veränderungen wie Hypertrophie, Polarisierung, Wanderung und Hochregulation bestimmter Intermediärfilamente. Diese Veränderungen werden insgesamt als „reaktive Gliose“ zusammengefasst. Darüber hinaus scheinen nach akuten Gehirnverletzungen einige reife, postmitotische Astrozyten in der Großhirnrinde zu de-differenzieren, in den Zellzyklus einzutreten und begrenzt Stammzelleigenschaften zu erlangen. Es ist bisher nicht bekannt, ob alle Astrozyten auf Verletzungen reagieren, oder ob Teilpopulationen verschiedener Plastizität existieren. Weiterhin sind die Mechanismen, die zum Anstieg von Astrozyten nach akuter Verletzung führen, z.B. Proliferation und Wanderung, in vivo bislang nicht verstanden. Deshalb wurde hier erstmals das Verhalten von Fluoreszenz-markierten Astrozyten im Gehirn der Maus nach akuter kortikaler Verletzung live über einen längeren Zeitraum mittels 2-Photonenmikroskopie untersucht. Die Beobachtungen zeigten morphologische Veränderungen und heterogene Verhaltensmuster reaktiver Astrozyten, d.h. hypertrophe, polarisierende und sich teilende Astrozyten in Abhängigkeit von der Läsionsgröße. Im Gegensatz zu in vitro und in situ Studien, sowie bekannter Motilität anderer Typen von Gliazellen, wurde die Wanderung von Astrozyten zum Ort der Verletzung in vivo nicht beobachtet. Allerdings wurde entdeckt, dass sich nur eine kleine Teilpopulation von Astrozyten teilt, und diese vorrangig in direktem Kontakt mit Blutgefäßen (juxtavaskulär) liegt. Selbst nach Verlust der RhoGTPase Cdc42 – einem Schlüsselfaktor für Zellpolarität –, der zu einem Proliferationsdefekt der Astrozyten führte, blieb die vaskuläre Nische erhalten. In einem in vitro Verletzungsmodell zeigten cdc42-defizienten Astrozyten Polaritätsdefekte, verbunden mit desorientierter Wanderung und verminderter Zellteilung. Schlussfolgernd, spielen Cdc42-vermittelte Signalwege eine wichtige Rolle für die Reaktion von Astrozyten auf eine akute Verletzung in vitro und in vivo. Die hier präsentierte Studie trägt bedeutend zum Verständnis reaktiver Astrozyten in Bezug auf deren Rolle in der Narbenbildung und Regeneration von geschädigtem Hirngewebe bei. Insbesondere wurden neue Erkenntnisse über verschiedene Teilpopulationen von Astrozyten mit vermutlich unterschiedlichen Funktionen gewonnen. Hierbei konnte vor allem den juxtavaskulär proliferierenden Astrozyten nach einer traumatischen Hirnverletzung hohe Plastizität zugesprochen werden

A Novel Validation Algorithm Allows for Automated Cell Tracking and the Extraction of Biologically Meaningful Parameters

Automated microscopy is currently the only method to non-invasively and label-free observe complex multi-cellular processes, such as cell migration, cell cycle, and cell differentiation. Extracting biological information from a time-series of micrographs requires each cell to be recognized and followed through sequential microscopic snapshots. Although recent attempts to automatize this process resulted in ever improving cell detection rates, manual identification of identical cells is still the most reliable technique. However, its tedious and subjective nature prevented tracking from becoming a standardized tool for the investigation of cell cultures. Here, we present a novel method to accomplish automated cell tracking with a reliability comparable to manual tracking. Previously, automated cell tracking could not rival the reliability of manual tracking because, in contrast to the human way of solving this task, none of the algorithms had an independent quality control mechanism; they missed validation. Thus, instead of trying to improve the cell detection or tracking rates, we proceeded from the idea to automatically inspect the tracking results and accept only those of high trustworthiness, while rejecting all other results. This validation algorithm works independently of the quality of cell detection and tracking through a systematic search for tracking errors. It is based only on very general assumptions about the spatiotemporal contiguity of cell paths. While traditional tracking often aims to yield genealogic information about single cells, the natural outcome of a validated cell tracking algorithm turns out to be a set of complete, but often unconnected cell paths, i.e. records of cells from mitosis to mitosis. This is a consequence of the fact that the validation algorithm takes complete paths as the unit of rejection/acceptance. The resulting set of complete paths can be used to automatically extract important biological parameters with high reliability and statistical significance. These include the distribution of life/cycle times and cell areas, as well as of the symmetry of cell divisions and motion analyses. The new algorithm thus allows for the quantification and parameterization of cell culture with unprecedented accuracy. To evaluate our validation algorithm, two large reference data sets were manually created. These data sets comprise more than 320,000 unstained adult pancreatic stem cells from rat, including 2592 mitotic events. The reference data sets specify every cell position and shape, and assign each cell to the correct branch of its genealogic tree. We provide these reference data sets for free use by others as a benchmark for the future improvement of automated tracking methods

Human keratinocytes have two interconvertible modes of proliferation.

Single stem cells, including those in human epidermis, have a remarkable ability to reconstitute tissues in vitro, but the cellular mechanisms that enable this are ill-defined. Here we used live imaging to track the outcome of thousands of divisions in clonal cultures of primary human epidermal keratinocytes. Two modes of proliferation were seen. In 'balanced' mode, similar proportions of proliferating and differentiating cells were generated, achieving the 'population asymmetry' that sustains epidermal homeostasis in vivo. In 'expanding' mode, an excess of cycling cells was produced, generating large expanding colonies. Cells in expanding mode switched their behaviour to balanced mode once local confluence was attained. However, when a confluent area was wounded in a scratch assay, cells near the scratch switched back to expanding mode until the defect was closed. We conclude that the ability of a single epidermal stem cell to reconstitute an epithelium is explained by two interconvertible modes of proliferation regulated by confluence.The initial association of holoclone and paraclone type behaviour in clonal cultures of NFSK with stem and balanced progenitor dynamics was due to BDS working in collaboration with PHJ, VN-N, David Doupé and Allon Klein, based on the quantitative analysis of published and unpublished colony size distributions6 . We thank Gözde Akdeniz & David Doupé for experimental work that led up to the project that was analysed by Allon Klein and Genneth Zhang, Patrick Lombard at the Wellcome TrustMedical Research Council Cambridge Stem Cell Institute for Bioinformatics analysis and Esther Choolun for technical assistance. We acknowledge the support of the Wellcome Trust, Cambridge Cancer Centre, Medical Research Council, the NC3Rs (National Centre for the Replacement, Refinement and Reduction of Animals in Research) and Cancer Research UK (Programme grant C609/A17257).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb328

Spatiotemporal Identification of Cell Divisions Using Symmetry Properties in Time-Lapse Phase Contrast Microscopy

A variety of biological and pharmaceutical studies, such as for anti-cancer drugs, require the quantification of cell responses over long periods of time. This is performed with time-lapse video microscopy that gives a long sequence of frames. For this purpose, phase contrast imaging is commonly used since it is minimally invasive. The cell responses of interest in this study are the mitotic cell divisions. Their manual measurements are tedious, subjective, and restrictive. This study introduces an automated method for these measurements. The method starts with preprocessing for restoration and reconstruction of the phase contrast time-lapse sequences. The data are first restored from intensity non-uniformities. Subsequently, the circular symmetry of the contour of the mitotic cells in phase contrast images is used by applying a Circle Hough Transform (CHT) to reconstruct the entire cells. The CHT is also enhanced with the ability to “vote” exclusively towards the center of curvature. The CHT image sequence is then registered for misplacements between successive frames. The sequence is subsequently processed to detect cell centroids in individual frames and use them as starting points to form spatiotemporal trajectories of cells along the positive as well as along the negative time directions, that is, anti-causally. The connectivities of different trajectories enhanced by the symmetry of the trajectories of the daughter cells provide as topological by-products the events of cell divisions together with the corresponding entries into mitoses as well as exits from cytokineses. The experiments use several experimental video sequences from three different cell lines with many cells undergoing mitoses and divisions. The quantitative validations of the results of the processing demonstrate the high performance and efficiency of the method

A computational framework for particle and whole cell tracking applied to a real biological dataset

Cell tracking is becoming increasingly important in cell biology as it provides a valuable tool for analysing experimental data and hence furthering our understanding of dynamic cellular phenomena. The advent of high-throughput, high-resolution microscopy and imaging techniques means that a wealth of large data is routinely generated in many laboratories. Due to the sheer magnitude of the data involved manual tracking is often cumbersome and the development of computer algorithms for automated cell tracking is thus highly desirable. In this work, we describe two approaches for automated cell tracking. Firstly, we consider particle tracking. We propose a few segmentation techniques for the detection of cells migrating in a non-uniform background, centroids of the segmented cells are then calculated and linked from frame to frame via a nearest-neighbour approach. Secondly, we consider the problem of whole cell tracking in which one wishes to reconstruct in time whole cell morphologies. Our approach is based on fitting a mathematical model to the experimental imaging data with the goal being that the physics encoded in the model is reflected in the reconstructed data. The resulting mathematical problem involves the optimal control of a phase-field formulation of a geometric evolution law. Efficient approximation of this challenging optimal control problem is achieved via advanced numerical methods for the solution of semilinear parabolic partial differential equations (PDEs) coupled with parallelisation and adaptive resolution techniques. Along with a detailed description of our algorithms, a number of simulation results are reported on. We focus on illustrating the effectivity of our approaches by applying the algorithms to the tracking of migrating cells in a dataset which reflects many of the challenges typically encountered in microscopy data

Investigating optimal time step intervals of imaging for data quality through a novel fully-automated cell tracking approach

Computer-based fully-automated cell tracking is becoming increasingly important in cell biology, since it provides unrivalled capacity and efficiency for the analysis of large datasets. However, automatic cell tracking’s lack of superior pattern recognition and error-handling capability compared to its human manual tracking counterpart inspired decades-long research. Enormous efforts have been made in developing advanced cell tracking packages and software algorithms. Typical research in this field focuses on dealing with existing data and finding a best solution. Here, we investigate a novel approach where the quality of data acquisition could help improve the accuracy of cell tracking algorithms and vice-versa. Generally speaking, when tracking cell movement, the more frequent the images are taken, the more accurate cells are tracked and, yet, issues such as damage to cells due to light intensity, overheating in equipment, as well as the size of the data prevent a constant data streaming. Hence, a trade-off between the frequency at which data images are collected and the accuracy of the cell tracking algorithms needs to be studied. In this paper, we look at the effects of different choices of the time step interval (i.e., the frequency of data acquisition) within the microscope to our existing cell tracking algorithms. We generate several experimental data sets where the true outcomes are known (i.e., the direction of cell migration) by either using an effective chemoattractant or employing no-chemoattractant. We specify a relatively short time step interval (i.e., 30 s) between pictures that are taken at the data generational stage, so that, later on, we may choose some portion of the images to produce datasets with different time step intervals, such as 1 min, 2 min, and so on. We evaluate the accuracy of our cell tracking algorithms to illustrate the effects of these different time step intervals. We establish that there exist certain relationships between the tracking accuracy and the time step interval associated with experimental microscope data acquisition. We perform fully-automatic adaptive cell tracking on multiple datasets, to identify optimal time step intervals for data acquisition, while at the same time demonstrating the performance of the computer cell tracking algorithms

Severe arterial injury heals with a complex clonal structure involving a large fraction of surviving smooth muscle cells.

BACKGROUND AND AIMS Smooth muscle cell (SMC) lineage cells in atherosclerosis and flow cessation-induced neointima are oligoclonal, being recruited from a tiny fraction of medial SMCs that modulate and proliferate. The present study aimed to investigate the clonal structure of SMC lineage cells healing more severe arterial injury. METHODS Arterial injury (wire, stretch, and partial ligation) was inflicted on the right carotid artery in mice with homozygous, SMC-restricted, stochastically recombining reporter transgenes that produced mosaic expression of 10 distinguishable fluorescent phenotypes for clonal tracking. Healed arteries and contra-lateral controls were analyzed after 3 weeks. Additional analysis of cell death and proliferation after injury was performed in wildtype mice. RESULTS The total number of SMC lineage cells in healed arteries was comparable to normal arteries but comprised significantly fewer fluorescent phenotypes. The population had a complex, intermixed, clonal structure. By statistical analysis of expected versus observed fractions of fluorescent phenotypes and visual inspection of coherent groups of same-colored cells, we concluded that >98% of SMC lineage cells in healed arteries belonged to a detectable clone, indicating that nearly all surviving SMCs after severe injury at some point undergo proliferation. This was consistent with serial observations in the first week after injury, which showed severe loss of medial cells followed by widespread proliferation. CONCLUSIONS After severe arterial injury, many surviving SMCs proliferate to repair the media and form a neointima. This indicates that the fraction of medial SMCs that are mobilized to repair arteries increases with the level of injury.This study was supported by grants from the Novo Nordisk Foundation (NNF17OC0030688 and NNF21OC0071830).S

Cell and Fibronectin Dynamics During Branching Morphogenesis

Branching morphogenesis is a dynamic developmental process shared by many organs, but the mechanisms that reorganize cells during branching morphogenesis are not well understood. We hypothesized that extensive cell rearrangements are involved, and investigated cell migration using two-color confocal time-lapse microscopy to image cell and extracellular-matrix dynamics in developing salivary glands. We labeled submandibular salivary gland (SMG) epithelial cells with green fluorescent protein and matrix with fluorescent fibronectin. Surprisingly, we observed substantial, rapid and relatively random migration of individual epithelial cells during branching morphogenesis. We predicted that cell migration would decrease after formation of acini and, indeed, found that rapid cell movements do not occur in SMG from newborn mice. However, in embryonic SMG epithelial cells, we observed an absence of choreographed cell migration, indicating that patterned cell migration alone cannot explain the highly ordered process of branching morphogenesis. We therefore hypothesized a role for directional fibronection assembly in branching. Washout and pulse-chase experiments revealed that older fibronectin accumulates at the base of the clefts and translocates inwards as a wedge, with newer fibronectin assembling behind it. These findings identify a new mechanism for branching morphogenesis involving directional fibronectin translocation superimposed on individual cell dynamics