Browsing by Subject "human"

In the past few years, there has been an increased consideration on the stem cell niche as a key factor to regulate stem cell maintenance and differentiation. Research on characterization of the stem cell microenvironment boosted after the determination of long-term three-dimensional (3D) tissue cultures, or so-called organoids. Organoids are derived from stem cells which self-organize in 3D multicellular structures upon embedding in an extracellular matrix mimic, such as Matrigel®. Their main advantage is these structures resemble the architectural distribution of the tissue of origin in vivo. Likewise, the cellular components of organoids vary depending on multiple variables as the tissue of origin and the growth factors they have access to. As a result of advances in this technique, some stem cell niches have been well characterized, as in the case of intestinal stem cells (ISCs), while others remain elusive as in case of the human gastric stem cells (hGSCs). Along with the remarkable development of 3D cultures, the interest of ECM proteins in stem cell regulation increased. Matrigel® is a rich matrix composed of several adhesive proteins such as laminins and collagens. Aside from providing structural support, the extracellular matrix (ECM) proteins forming this matrix contribute to cell adhesion and signalling. However, Matrigel® composition cannot be modified or even well-characterized due to its origin from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Additionally, it has been demonstrated that contains a high batch-to-batch variability. Other techniques to study the effects of individual ECM proteins have been used such as coating of tissue culture plates with ECM proteins. However, the biomechanical properties in this model are far from being physiological. Therefore, although preliminary results can be obtained using this technique, results extrapolation to an in vivo model can be questioned. To date, there is a lack of a reproducible, high-throughput and reliable technique to test the effect of ECM proteins on human gastric stem cells behavior. This Master’s thesis presents a novel transwell device containing a polyethylene glycol (PEG)-based hydrogel enriched with human ECM proteins to test their effect on human gastric stem cell regulation. Preliminary results showed that gastric organoid-derived epithelial cells (GODE) grown on hydrogels with ECM proteins that are localized at base of the gastric glands, such as Laminin-211, had a higher stem cell marker expression than the control grown on ECM proteins that are uniformly localized in vivo. Additionally, when GODE were grown on hydrogels containing ECM proteins that are localized at the surface of the native gastric epithelium, expression of surface gastric mucins markers was enhanced. These preliminary results highlight the utility of the optimized transwell device to further shed light on how the human gastric stem cells are regulated and what is the effect of the ECM proteins surrounding them.

Drug-induced liver injury (DILI) is a relatively rare hepatic condition that can be classified as predictable and unpredictable. However, DILI is a primary reason for drug withdrawals, post-marketing warnings, and restrictions of use. DILI is a problem for the drug users but also for the pharmaceutical industry and regulatory bodies. From the perspective of patients' and clinicians', DILI is the major cause of acute liver injury. At present, a major problem predicting DILI in drug discovery is a poor understanding of its mechanisms as well as the complexity of DILI pathogenicity. The main mechanism behind DILI are alterations in bile acid homeostasis, oxidative stress, and mitochondrial dysfunction. More than 50 % of drugs causing DILI are causing mitochondrial impairment. If the normal function of mitochondria is disturbed, the energy production of the cell decreases, and cell function decline leading eventually to the cell death. In this study prediction of mitochondrial toxicity was studied using cryopreserved primary hepatocytes of humans and rats. The aim of the study was to clarify if there are interspecies differences in the prediction of toxicity but also investigate possible differences in the mechanisms behind hepatotoxicity by using three well-known compounds toxic to mitochondria. To determine these differences, total cellular ATP was measured after 2- and 24- hour exposure time to gain information on overall viability and possible adaptive responses. Mitochondrial energy pathways were studied as a real-time monitoring acute exposure of test compounds. Morphology, location, and possible adaptive response of mitochondria were studied using a fluorescent probe and antibody staining combined with high content imaging (HCI). Overall, primary rat hepatocytes were more sensitive to the test compounds than human hepatocytes. Also, there were differences between human hepatocyte batches that may reflect the metabolic differences between hepatocyte donors. Immunolabeling did not bring any additional values compared to the fluorescent probe staining in the study of morphology of mitochondria. Additionally, it was noticed that treatment with paraformaldehyde significantly changed the hepatocyte mitochondria morphology. Overall, more effort is needed to develop image analysis of mitochondria morphology. Finally, studying mitochondrial morphology has proven to be difficult, and this study did not unfortunately reveal any information about the adaptive responses of mitochondria for drug-induced liver injury.