Characterization of Embryonic Stem (ES) Neuronal Differentiation Combining Atomic Force, Confocal and DIC Microscopy Imaging

The ultimate clinical implementation of embryonic stem cells will require methods and protocols to turn these unspecialized cells into the fully functioning cell types found in a wide variety of tissues and organs. In order to achieve this, it is necessary to clearly understand the signals and cues that direct embryonic stem cell differentiation. This book provides a snapshot of current research on the differentiation of embryonic stem cells to a wide variety of cell types, including neural, cardiac, endothelial, osteogenic, and hepatic cells. In addition, induced pluripotent stem cells and other pluripotent stem cell sources are described. The book will serve as a valuable resource for engineers, scientists, and clinicians as well as students in a wide range of disciplines

DNA methyltransferase-3-dependent nonrandom template segregation in differentiating embryonic stem cells.

Asymmetry of cell fate is one fundamental property of stem cells, in which one daughter cell self-renews, whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms, such as plants, fungi, and mammals, has already been shown. However, before this current work, asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs), and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing, differentiating human and mouse ESCs. Moreover, we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3), indicating a molecular mechanism that regulates this phenomenon. Furthermore, our data support the hypothesis that retention of chromatids with the old template DNA preserves the epigenetic memory of cell fate, whereas localization of new DNA strands and de novo DNA methyltransferase to the lineage-destined daughter cell facilitates epigenetic adaptation to a new cell fate

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

Remote-refocusing light-sheet fluorescence microscopy enables 3D imaging of electromechanical coupling of hiPSC-derived and adult cardiomyocytes in co-culture

Improving cardiac function through stem-cell regenerative therapy requires functional and structural integration of the transplanted cells with the host tissue. Visualizing the electromechanical interaction between native and graft cells necessitates 3D imaging with high spatio-temporal resolution and low photo-toxicity. A custom light-sheet fluorescence microscope was used for volumetric imaging of calcium dynamics in co-cultures of adult rat left ventricle cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes. Aberration-free remote refocus of the detection plane synchronously to the scanning of the light sheet along the detection axis enabled fast dual-channel 3D imaging at subcellular resolution without mechanical sample disturbance at up to 8 Hz over a ∼300 µm × 40 µm × 50 µm volume. The two cell types were found to undergo electrically stimulated and spontaneous synchronized calcium transients and contraction. Electromechanical coupling improved with co-culture duration, with 50% of adult-CM coupled after 24 h of co-culture, compared to 19% after 4 h (p = 0.0305). Immobilization with para-nitroblebbistatin did not prevent calcium transient synchronization, with 35% and 36% adult-CM coupled in control and treated samples respectively (p = 0.91), indicating that electrical coupling can be maintained independently of mechanotransduction

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

3D imaging and quantitative analysis of intact tissues and organs

Embryonic development and tumor growth are highly complex and dynamic processes that exist in both time and space. To fully understand the molecular mechanisms that control these processes, it is crucial to study RNA expression and protein translation with single-cell spatiotemporal resolution. This is feasible by microscopic imaging that enables multidimensional assessments of cells, tissues, and organs. Here, a time-lapse calcium imaging and three-dimensional imaging was used to study physiological development of the brain or pathological development of cancer, respectively. In Paper I, spatiotemporal calcium imaging revealed a new mechanism of neurogenesis during brain development. In Paper II, a new clearing method of clinically stored specimens, DIPCO (diagnosing immunolabeled paraffin-embedded cleared organs), was developed that allows better characterization and staging of intact human tumors. In Paper III, the DIPCO method was applied to determine tumor stage and characterize the microlymphatic system in bladder cancer. In Paper IV, a novel method for RNA labeling of volumetric specimens, DIIFCO (diagnosing in situ and immunofluorescence-labeled cleared onco-sample) was developed to study RNAs expression and localization in intact tumors. Overall, the aim of the thesis was to demonstrate that multidimensional imaging extends the understanding of both physiological and pathological biological developmental processes