Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools

Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant software, a commercially available video bioinformatics tool, can be used to extract quantitative data on: (1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and (4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and colonies. Time-lapse experiments in which different cell types were subjected to various chemical exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels). Colony and single cell migration were studied by measuring total displacement (distance between the starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production, cells were pre-loaded with MitoSOX Red™, a mitochondrial ROS (superoxide) indicator, treated with various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant software can be used to evaluate biological processes in living cells, and the protocols developed in this project can be applied to basic research and toxicological studies, or to monitor quality control in culture facilities

Methods for Spatio-Temporal Analysis of Embryo Cleavage In Vitro

Automated or semiautomated time-lapse analysis of early stage embryo images during the cleavage stage can give insight into the timing of mitosis, regularity of both division timing and pattern, as well as cell lineage. Simultaneous monitoring of molecular processes enables the study of connections between genetic expression and cell physiology and development. The study of live embryos poses not only new requirements on the hardware and embryo-holding equipment but also indirectly on analytical software and data analysis as four-dimensional video sequencing of embryos easily creates high quantities of data. The ability to continuously film and automatically analyze growing embryos gives new insights into temporal embryo development by studying morphokinetics as well as morphology. Until recently, this was not possible unless by a tedious manual process. In recent years, several methods have been developed that enable this dynamic monitoring of live embryos. Here we describe three methods with variations in hardware and software analysis and give examples of the outcomes. Together, these methods open a window to new information in developmental embryology, as embryo division pattern and lineage are studied in vivo

Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Background: One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. Methodology/Principal Findings: We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. Conclusions/Significance: The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development

Time-Lapse Microscopy

Time-lapse microscopy is a powerful, versatile and constantly developing tool for real-time imaging of living cells. This review outlines the advances of time-lapse microscopy and refers to the most interesting reports, thus pointing at the fact that the modern biology and medicine are entering the thrilling and promising age of molecular cinematography

Automating assessment of human embryo images and time-lapse sequences for IVF treatment

As the number of couples using In Vitro Fertilization (IVF) treatment to give birth increases, so too does the need for robust tools to assist embryologists in selecting the highest quality embryos for implantation. Quality scores assigned to embryonic structures are critical markers for predicting implantation potential of human blastocyst-stage embryos. Timing at which embryos reach certain cell and development stages in vitro also provides valuable information about their development progress and potential to become a positive pregnancy. The current workflow of grading blastocysts by visual assessment is susceptible to subjectivity between embryologists. Visually verifying when embryo cell stage increases is tedious and confirming onset of later development stages is also prone to subjective assessment. This thesis proposes methods to automate embryo image and time-lapse sequence assessment to provide objective evaluation of blastocyst structure quality, cell counting, and timing of development stages

Time-lapse embryo imaging and morphokinetic profiling: towards a general characterisation of embryogenesis

In vitro fertilisation is an effective method of assisted reproductive technology in both humans and certain non-human animal species. In most species, specifically, in humans and livestock, high in vitro fertilisation success rates are achieved via the transfer of embryos with the highest implantation and subsequent developmental potential. In order to reduce the risk of multiple gestation, which could be a result of the transfer of several embryos per cycle, restrictive transfer policies and methods to improve single embryo selection have been implemented. A non-invasive alternative to standard microscopic observation of post-fertilisation embryo morphology and development is time-lapse technology; this enables continuous, uninterrupted observation of embryo development from fertilisation to transfer. Today, there are several time-lapse devices that are commercially available for clinical use, and methods in which time-lapse could be used to improve embryology are continually being assessed. Here we review the use of time-lapse technology in the characterisation of embryogenesis and its role in embryo selection. Furthermore, the prospect of using this technology to identify aneuploidy in human embryos, as well as the use of time-lapse to improve embryological procedures in agriculturally important species such as the pig and cow are discussed

An Engineered Approach to Stem Cell Culture: Automating the Decision Process for Real-Time Adaptive Subculture of Stem Cells

Current cell culture practices are dependent upon human operators and remain laborious and highly subjective, resulting in large variations and inconsistent outcomes, especially when using visual assessments of cell confluency to determine the appropriate time to subculture cells. Although efforts to automate cell culture with robotic systems are underway, the majority of such systems still require human intervention to determine when to subculture. Thus, it is necessary to accurately and objectively determine the appropriate time for cell passaging. Optimal stem cell culturing that maintains cell pluripotency while maximizing cell yields will be especially important for efficient, cost-effective stem cell-based therapies. Toward this goal we developed a real-time computer vision-based system that monitors the degree of cell confluency with a precision of 0.791±0.031 and recall of 0.559±0.043. The system consists of an automated phase-contrast time-lapse microscope and a server. Multiple dishes are sequentially imaged and the data is uploaded to the server that performs computer vision processing, predicts when cells will exceed a pre-defined threshold for optimal cell confluency, and provides a Web-based interface for remote cell culture monitoring. Human operators are also notified via text messaging and e-mail 4 hours prior to reaching this threshold and immediately upon reaching this threshold. This system was successfully used to direct the expansion of a paradigm stem cell population, C2C12 cells. Computer-directed and human-directed control subcultures required 3 serial cultures to achieve the theoretical target cell yield of 50 million C2C12 cells and showed no difference for myogenic and osteogenic differentiation. This automated vision-based system has potential as a tool toward adaptive real-time control of subculturing, cell culture optimization and quality assurance/quality control, and it could be integrated with current and developing robotic cell cultures systems to achieve complete automation

Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models.

To-date, most invasion or migration assays use a modified Boyden chamber-like design to assess migration as single-cell or scratch assays on coated or uncoated planar plastic surfaces. Here, we describe a 96-well microplate-based, high-content, three-dimensional cell culture assay capable of assessing invasion dynamics and molecular signatures thereof. On applying our invasion assay, we were able to demonstrate significant effects on the invasion capacity of fibroblast cell lines, as well as primary lung fibroblasts. Administration of epidermal growth factor resulted in a substantial increase of cellular invasion, thus making this technique suitable for high-throughput pharmacological screening of novel compounds regulating invasive and migratory pathways of primary cells. Our assay also correlates cellular invasiveness to molecular events. Thus, we argue of having developed a powerful and versatile toolbox for an extensive profiling of invasive cells in a 96-well format. This will have a major impact on research in disease areas like fibrosis, metastatic cancers, or chronic inflammatory states

Regulation of Early Zebrafish Embryogenesis by Calcium Signaling and Dachsous1b Cadherin

Early animal embryogenesis entails a dynamic combination of embryonic cleavages, axial patterning, and gastrulation movements to shape a basic body plan. The underlying molecular signaling responsible for regulating this process remains poorly understood. In this thesis work, I first review recent progress in understanding of gastrulation movements in various model organisms brought by advances in imaging techniques. The externally developing and optically translucent zebrafish embryo is an ideal model organism to study vertebrate embryonic development by in vivo imaging. The objective of my thesis research is to leverage experimental advantages in the zebrafish model to uncover novel regulators and elucidate the molecular mechanisms involved in early vertebrate embryogenesis. Calcium signaling has been implicated in the control of many aspects of embryonic development. However, the spatiotemporal dynamics of calcium signaling during embryogenesis are not well characterized. By generating stable transgenic zebrafish lines ubiquitously expressing GCaMP6s, a genetically encoded calcium indicator, I demonstrated higher activities of calcium signaling during cleavage and blastula stages compared to previous reports. In addition, I showed that superficial dorsal-biased calcium signaling during blastula and gastrula stages was strongly correlated with and dependent on the dorsal organizer establishment. In the developing gastrulae, I directly visualized calcium activity in the dorsal forerunner cells and showed it was modulated by Nodal signaling in a cell non-autonomous manner. The GCaMP6s transgenic lines revealed with unprecedented spatiotemporal resolution the dynamic calcium signaling during early zebrafish embryogenesis and provide a superior tool for future studies. In zebrafish, mutations in atypical cadherin dachsous1b/dchs1b cause pleiotropic embryonic defects, including abnormal cleavages. Using the GCaMP6s transgenic reporter to examine the furrow-associated calcium activity in zebrafish dchs1b mutants, I showed that abnormal cleavages in dchs1b mutants were due to furrow progression defects during cytokinesis. These defects were likely caused by misregulated microtubules, as in vivo imaging of fluorescently marked microtubules during cleavage stages revealed reduced microtubule dynamics and impaired midzone microtubule assembly in dchs1b mutants. I further identified Ttc28 cytoplasmic protein as a molecular link between Dchs1b and microtubule dynamics. My biochemical experiments revealed that Dchs1b physically interacts via its intracellular domain with the tetratricopeptide repeat domain of Ttc28, and controls its subcellular distribution. Moreover, genetic inactivation of ttc28 resulted in increased microtubule dynamics and suppressed the microtubule defects in dchs1b mutants, suggesting a mechanism through which Dchs1b controls embryonic cleavages. In the last part of my thesis, I aimed to determine whether the chemokine ligand Ccl19.a1, a potential upstream regulator of calcium signaling, is required for axial patterning in zebrafish. I demonstrated that TALEN-generated ccl19a.1 mutations produce mildly dorsalized phenotypes and partially suppress the ventralized ichabod/ctnnb2 mutant phenotypes to influence axis formation, providing a genetic evidence for Ccl19.1 acting as a negative regulator of β-catenin and axis formation. Together, my work make several advances in understanding early vertebrate embryogenesis: it characterizes dynamic calcium signaling during zebrafish embryogenesis with a superior spatiotemporal resolution, reveals that Dchs1b regulates microtubule dynamics and embryonic cleavages by interacting with Ttc28 and regulating its subcellular distribution, and provides genetic evidence that Ccl19a.1 is necessary to limit β-catenin activity and consequently axis formation in zebrafish