Analysis of glioma cell heterogeneity by lineage-tracing in murine and human model systems

Intra-tumour heterogeneity and plasticity are key factors in treatment resistance and the recurrence of Glioblastoma (GBM), which is invariably fatal. Genetics, epigenetics, cell metabolism and plastic cancer stem cell (CSC) hierarchies interact with volatile micro-environmental forces to promote and shape cell identity. Improved understanding of these factors will inform more precise and effective GBM therapies. Here, we aim to develop a fluorescent tracking approach for patient derived GBM cells to investigate the relationship between clones, environment and CSC marker expression. Using a murine GBM model combined with Rosa26-confetti fluorescent labelling, we trialled suitable techniques for detection of labelled tumour clones and concluded fluorescent imaging and flow cytometry were the most effective. For patient-derived cells, we modified LeGO-vector fluorescent labelling with the aim of tracking a greater number of clones. We further optimised this technique for simultaneous flow cytometry detection of clones and their CSC marker expression. In the final chapter, we address the hypothesis that whole population CSC surface marker plasticity is a result of emergent clonal predominance. In two patient derived GBM lines, under steady-state environmental conditions, serial passaging and assessment of clonal marker expression detected distinct marker expression patterns between clones in the same culture dish. For both cell lines, transfer and culture of clonal mixtures to Matrigel® spheroids produced an expected plastic transition in population marker expression but also considerable predominance of certain clones. While the clonalsurface marker dynamics of the two cell lines were markedly distinct, divergent surface marker plasticity between clones of the same cell line was a consistent observation. Taken together these results supported our hypothesis that population marker plasticity is in part a result of emergent clonal predominance. We propose our developed techniques are suitable for rapid and economic characterisation of patient specific gene disruption, therapeutic vulnerabilities and resistance mechanisms

Tight Regulation of Mechanotransducer Proteins Distinguishes the Response of Adult Multipotent Mesenchymal Cells on PBCE-Derivative Polymer Films with Different Hydrophilicity and Stiffness

: Mechanotransduction is a molecular process by which cells translate physical stimuli exerted by the external environment into biochemical pathways to orchestrate the cellular shape and function. Even with the advancements in the field, the molecular events leading to the signal cascade are still unclear. The current biotechnology of tissue engineering offers the opportunity to study in vitro the effect of the physical stimuli exerted by biomaterial on stem cells and the mechanotransduction pathway involved in the process. Here, we cultured multipotent human mesenchymal/stromal cells (hMSCs) isolated from bone marrow (hBM-MSCs) and adipose tissue (hASCs) on films of poly(butylene 1,4-cyclohexane dicarboxylate) (PBCE) and a PBCE-based copolymer containing 50 mol% of butylene diglycolate co-units (BDG50), to intentionally tune the surface hydrophilicity and the stiffness (PBCE = 560 Mpa; BDG50 = 94 MPa). We demonstrated the activated distinctive mechanotransduction pathways, resulting in the acquisition of an elongated shape in hBM-MSCs on the BDG50 film and in maintaining the canonical morphology on the PBCE film. Notably, hASCs acquired a new, elongated morphology on both the PBCE and BDG50 films. We found that these events were mainly due to the differences in the expression of Cofilin1, Vimentin, Filamin A, and Talin, which established highly sensitive machinery by which, rather than hASCs, hBM-MSCs distinguished PBCE from BDG50 films

Unraveling nanoscale alterations in liver cell fenestrations - Morphological studies via optical super-resolution microscopy approaches

The endothelium makes up the innermost cell layer of blood vessels. It consists of a thin layer of simple squamous cells, forming an interface between circulating blood and the surrounding tissue. Endothelial cells of different vascular beds are specialized according to tissue-specific functions. For this project emphasis was placed upon high-resolution methods enabling the study of liver sinusoidal endothelial cells (LSECs) below the diffraction limit of visible light (~200 nm). LSECs have unusual morphology with as much of 20% of their surface covered with cellular fenestrations - holes through the cells of 50-300 nm diameter. These allow bi-directional flow of plasma from the sinusoids to the surrounding hepatocytes, while retaining blood cells in the sinusoidal lumen. Little is known about the function of fenestrations, their regulation, and their role in the transfer of metabolites, viruses, lipoproteins and pharmaceuticals to other cells of the liver. There are two major challenges with the study of LSEC fenestrations; i) the majority have diameters smaller than the diffraction limit of visible light and; ii) they disappear rapidly in cultured LSEC, and there are no cell line alternatives that express fenestrations. To address the first challenge, the project used classical super resolution imaging technologies such as scanning electron microscopy, and two novel super-resolution optical microscopy modalities: dSTORM (direct stochastic optical reconstruction microscopy) and SIM (structured illumination microscopy) to study the in vitro effects of xanthines, sildenafil and oxidized LDL on LSEC fenestrations. One of the xanthines, theobromine, and sildenafil increased both the frequency and diameter of fenestrations in cultured LSEC. While oxidized LDL caused major disruptions in LSEC fenestration morphology. Finally, to address the second challenge, namely the rapid loss of fenestrations in LSEC, a cryopreservation method for freshly isolated LSEC was developed such that they can be used at researchers’ convenience, rather than directly after isolation from liv

Development and applications of CRISPR-based functional genomics platforms in human iPSC-derived neurons

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells such as neurons. Neurons possess unique cell biological properties that enable them to exert highly specialized physiological functions. To enable systematic characterization of neuronal cell biology, we established CRISPR interference (CRISPRi)- and CRISPR activation (CRISPRa)-based platforms in human neurons derived from induced pluripotent stem cells (iPSCs) that allow robust repression or activation of endogenous genes and large-scale genetic screens in human neurons. Using this toolkit, we performed multiple genome-wide screens to identity genes controlling neuronal survival under unstressed and oxidative stress conditions and genes regulating neuronal redox homeostasis. These screens provide numerous novel biological insights. We also demonstrate that our platforms can be coupled with single-cell RNA sequencing and longitudinal high-content imaging to reveal consequences of genetic perturbations on gene expression and neuronal morphology respectively. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons

Study of the materno-fetal microchimerism of the APC using MHCII/EGFP mouse model and clearing histological techniques

Microchimerism arises from the exchange of cells between genetically distinct individuals. The coexistence of genetically distinct cell populations within a single organism has possible effects on health and functioning of individuals immune systems, but the exact mechanisms of action are often not yet known. With the development of microscopic technologies and software for data analysis, the possibilities of detection and phenotyping of these rare cell populations are expanding. My intention in this work is to find maternal microchimerism in embryonic tissues (E13) and intestines of breastfed pups using MHCII/EGFP knock-in mouse model. Several different technologies potentially suitable for the detection of maternal microchimeric cells in offspring tissues (light sheet fluorescent microscopy - LSFM, virtual slide microscopy and flow cytometry) were selected. Advanced analysis of the obtained samples from the light sheet microscopy using the creation of a neural network was used here. The presence of maternal microchimerism was not demonstrated by flow cytometry. Using LSFM, image data were obtained from intestinal samples of suckling pups, which were processed by the neural network method. Data analysis of embryos (E13) obtained by the same method did not allow data analysis due to high...Mikrochimérismus vzniká výměnou buněk mezi geneticky odlišnými jedinci. Koexistence geneticky odlišných populací buněk v rámci jednoho organismu přináší možné dopady na zdraví a fungování imunitního systému jedinců, ale přesné mechanismy působení nejsou ještě často známy. S rozvojem mikroskopických technologií a softwarů pro analýzu dat se rozšiřují možnosti detekce a fenotypizace těchto vzácných buněčných populací. Mým záměrem v této práci je vyhledání maternálního mikrochimérismu v tkáních embrya a střevech kojených mláďat s využitím MHCII/EGFP knock-in myšího modelu, který jsme k tomuto účelu vytvořili. Bylo vybráno několik různých technologií potenciálně vhodných pro detekci maternálních mikrochimérních buněk v tkáních potomka (fluorescenční light sheet mikroskopie - LSFM, virtual slide mikroskopie a průtoková cytometrie). Byla zde využita pokročilá analýza získaných vzorků z light sheet mikroskopie pomocí tvorby neuronové sítě. Metodou průtokové cytometrie přítomnost maternálního mikrochimérismu nebyla prokázána. Pomocí LSFM byla získána obrazová data ze vzorků střev kojených mláďat, která byla zpracována metodou neuronové sítě. U obrazových dat embryí (E13) získaných stejnou metodou nebyla možná analýza dat vlivem vysoké autofluorescence. Pro porovnání byla použita metoda virtual slide...Department of Cell BiologyKatedra buněčné biologiePřírodovědecká fakultaFaculty of Scienc

Development and application of electron microscopy methods for endocytic-secretory pathway studies in cells

Electron microscopy (EM) and tomography provides the most detailed view of the cellular ultrastructure and can be further extended to the structural level using cryogenic electron tomography. Fluorescent microscopy (FM) is used to complement this data with molecular identity information in a wide range of correlative microscopy techniques. In this thesis I present my work on the development of the correlative microscopy methods as well as the application of new and established electron microscopy methods to the study of the endocytic and secretory pathways. I developed a sample parallelization approach based on the fluorescent barcoding of budding yeast cells that allows high-throughput screening of yeast mutants using EM. Cells from different strains or under different conditions are grown in parallel and then subjected to combinatorial labeling with fluorescent dyes. Labeled cells are mixed together to generate a single sample which is subjected to high-pressure freezing, freeze-substitution and sectioning. The sections are imaged with FM and EM. FM data is used to determine the fluorescent barcode of each cell and thus its strain identity or experimental conditions, and high-resolution EM data can be collected in parallel for each of the strains or conditions. The total time spent on embedding and sectioning can be reduced up to 30 times using the developed protocol. I demonstrate the utility of the method by analyzing the variation of total multivesicular body volume (MVB) in different yeast strains. As a part of the collaborative project investigating the role of the ATPase Vps4 in the formation of MVBs, I performed correlative FM and electron tomography of MVBs containing Vps4. It showed that MVBs correlating with the Vps4 signal usually form clusters of more than one organelle and that the Vps4 signal correlates with MVBs actively forming intraluminal vesicles. Finally, I used subtomogram averaging to determine the COPI coat structure in situ, within its native cellular environment. I analyzed a tomographic dataset of cryo-lamella prepared by collaborators using focused ion beam milling of vitrified Chlamydomonas reinhardtii cells. I determined the COPI coat structure de novo and analyzed its variability during uncoating and within the Golgi stack. The COPI coat preserved its structure and stoichiometry during uncoating and in different Golgi regions. However the density of bound dilysine cargo and membrane thickness varied along the stack. In this thesis I have applied different EM methods to investigate morphological and structural aspects of the endocytic-secretory pathway. In the future such an integrative EM approach, ranging from functional genetic screens to structure determination, can be used to address multiple questions in cell biology