Video Bioinformatics: Human Embryonic Stem Cell Analysis With Machine Learning

Human Embryonic Stem Cell (hESC) have a great potential for regenerative medicine to provide treatments for Parkinson’s disease, Huntington’s disease, Type 1 diabetes mellitus, etc. Consequently, hESC are often used as a model in the biological assay to study the effects of chemical agents on the human body. Video analysis plays an important role for biological assays in the field of prenatal toxicology and stem cell differentiation. This thesis introduces machine learning techniques for detection, segmentation and classification for hESC analysis. For the detection, a bio-driven algorithm was used to detect cell regions in hESC images. Cell region detection is essential in stem cell focused analysis. It can prevent background information from contaminating the analysis and put more emphasis on processing the cell region. For the segmentation part, a bio-inspired method was proposed for bleb extraction and analysis over time. Bleb formation is a strong health indicator of the stem cell undergoing chemical reactions. Therefore, it is significant to biologist to analyze the formation process over time. For the classification, a deep learning structure was built with both labeled and unlabeled hESC data to classify the six common classes in stem cell images. The six classes are: 1). cell clusters, 2). debris, 3). unattached cells, 4). attached cells, 5). dynamically blebbing cells, and 6). apoptotically blebbing cells. Various results are provided on real video datasets collected using a phase contrast microscope and a Nikon Bio-station

Live analysis and function of Myc-mediated cell competition in mouse pluripotent stem cells

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 07-07-2017In the early mouse embryo and in embryonic stem cell (ESC) cultures the transcription factor Myc exhibits a cell-to-cell heterogeneous pattern. Cells expressing low levels of Myc are eliminated from the population by cell competition. Myc has been reported to promote cell reprograming to pluripotency and to regulate cell anabolism and proliferation in ESCs; however, the biological role of Mycdependent endogenous cell competition and the dynamics and regulation of Myc during this process remain unknown. Here we develop a new image analysis tool that allows us to track the temporal evolution of endogenous Myc levels, perform neighbourhood analysis in ESC cultures and generate 3D+t computerized data. We show that despite Myc degradation and resynthesis during mitosis, Myc levels are mostly heritable in ESC lineages. Cell competition results from random interactions between cells with high discrepancies in Myc levels. Myc-low cells (“losers”) temporally integrate contacts with Myc-high cells (“winners”), which leads to a progressive decrease in their own Myc levels until dying. Interestingly, endogenous Myc levels correlate with the pluripotency status; differentiation-primed cells express low Myc levels and are outcompeted by Myc-high naive ESCs. Indeed, cell competition inhibition results in an accumulation of primed cells. These observations in ESCs correlate with findings in the mouse epiblast. Moreover, we show that Myc levels directly determine the competitive ability of ESCs irrespective of the pluripotency status. Our results identify Myc as a mediator between differentiation status and competitive ability of pluripotent cells. Myc-driven endogenous cell competition thus acts as a mechanism to protect pluripotent stem cell pools from differentiation.Tanto en el embrión temprano de ratón como en cultivos de células madre embrionarias (CMEs), el factor de transcripción Myc muestra un patrón de expresión heterogéneo. Las células que expresan menos niveles de Myc son eliminadas de la población mediante competición celular. Se ha demostrado que Myc promueve la programación celular hacia un estado de pluripotencia y regula el anabolismo y la proliferación en CMEs; sin embargo, la función biológica de la competición celular endógena dependiente de Myc, así como la regulación y dinámica de la expresión este gen durante este proceso aún se desconocen. En esta tesis desarrollamos una novedosa herramienta de análisis de imagen, a partir de la cual realizamos análisis de células individuales y sus vecindarios y generamos un sistema de datos 3D+t computarizados. De esta manera demostramos que, a pesar de que Myc se degrada y se vuelve a sintetizar rápidamente durante la mitosis, sus niveles de expresión son en su mayor parte heredables. Además, la competición celular se desencadena por interacciones aleatorias entre células con grandes diferencias en sus niveles de Myc; de forma que las células que poseen bajos niveles de Myc (“perdedoras”) integran en el tiempo contactos con células con mayor expresión (“ganadoras”) lo que lleva a una reducción progresiva en sus propios niveles hasta que mueren. Cabe resaltar que los niveles endógenos de Myc correlacionan con el estado de pluripotencia; las células en un estado más avanzado de diferenciación expresan menos Myc siendo eliminadas por células más indiferenciadas y con altos niveles de expresión. De hecho, la inhibición de la competición celular produce una acumulación de células en proceso de diferenciación. Estas observaciones correlacionan con resultados obtenidos en estudios realizados en epiblasto de ratón. Además, demostramos que la eficiencia competitiva viene directamente determinada por los niveles de Myc independientemente del estado de pluripotencia. Estos resultados señalan a Myc como un intermediario entre el estado de diferenciación y la capacidad competitiva de las células pluripotentes. Por lo tanto, la competición celular endógena promovida por Myc funciona como un mecanismo de protección de pob0laciones de células madre pluripotentes frente a la diferenciación

METABOLIC HETEROGENEITY IN ISCHEMIA REPERFUSION INJURY: THE INSIDE STORY

It is well established that ischemia reperfusion (IR) injury can lead to life-threatening arrhythmias. Our group introduced the theory of metabolic sinks as a novel cause of arrhythmogenesis in a heart exposed to IR injury. Metabolic sinks are clusters of myocytes in which ischemia-induced reduction of ATP:ADP ratio increases open probability of sarcolemmal ATP-sensitive K+ channels. This in turn leads to hyperpolarization of membrane potential that reduces or ablates electrical excitability within the affected region of myocardium. To date, studies of metabolic sinks in intact hearts have been largely limited to use of fluorescent indicators to image mitochondrial membrane potential on the epicardial surface. This has revealed the existence of spatio-temporally evolving regions of myocardium within which mitochondrial membrane potential is depolarized, and whose presence influences electrical conduction and action potential duration. In order to explore the three-dimensional structure of metabolic sinks within the myocardium, we have developed a protocol for labeling an intact guinea pig heart exposed to IR injury and imaging any portion of the labeled heart using a custom designed automated volume imaging microtome (AVIM) to overcome the limited imaging field of laser scanning microscopy (LSM). Our AVIM is composed of low-cost components that can be easily installed and removed from a shared microscope. We have developed an open-source software signal processing pipeline to correct for imaging artifacts inherent to LSM and effectively reconstruct the acquired image volumes. Using this approach, we show that hearts undergoing reperfusion arrhythmias have an endocardium containing mostly depolarized mitochondria, with an abrupt transition to repolarized mitochondria in mid-myocardial to epicardial regions. Hearts not exhibiting reperfusion arrhythmias show a much more uniform distribution of depolarized mitochondria as a function of transmural location. These results show different stereotypical patterns of mitochondrial depolarization that are correlated with the presence and absence of reperfusion arrhythmias