Browsing by Subject "extracellular matrix"
Now showing items 1-5 of 5
-
(2022)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.
-
(2019)Despite the advances in the management of breast cancer, discovery of novel and targeted drugs remains a challenge. It has been suggested that drug failure rates in clinical trials might be diminished by improving the predictive potential of preclinical cancer models. Three-dimensional (3D) scaffold-based cell culture has emerged as an attractive platform for mimicking tissue-like microenvironment, since it is well-known that cells respond to the cues in the extracellular matrix (ECM). The aim of this thesis was to develop fibrin-based hydrogels and evaluate their performance in 3D cell culture of breast cancer cells. The fibrin gel formulation was first optimized by testing the effect of different buffers on gel properties. Structural properties were examined with scanning electron microscopy and mechanical properties measured with oscillatory rheometry. Three different fibrin concentrations of the optimized formulation were then used as scaffolds for DU4475 breast cancer cells. After seven days of culture, the morphology, phenotype and proliferation of the resulting cell structures were assessed by using techniques such as light microscopy, immunofluorescent confocal microscopy and Western blot analysis. The desired properties for 3D cell culture were obtained by preparing fibrin gels at high pH in the absence of calcium. The main finding of the thesis was that fibrin concentration strongly affected the phenotype of DU4475 cells, with cells cultured in the softest gel retaining their original characteristics to the greatest extent. In the future, the developed scaffold could possibly be used in drug discovery and personalized medicine by culturing tumor explants from patients. However, the methods used in the study must be further optimized and the results validated with other breast cancer cell lines and with primary tissues.
-
(2012)Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have two unique properties: the self-renewal capacity and the broad developmental potential. They both have their advantages and disadvantages, but the current perception is that hESCs and hiPSCs complement rather than replace each other. New scientific problems and ethical challenges will arise because stem cell research is developing rapidly. The potential of hiPSC and hESC technologies in drug discovery is tremendous. The human pluripotent stem cell (hPSC)-derived cells have a potential to replace a part of the current preclinical toxicity and efficacy screening tests and to prevent misrouted drug development and use for lead optimization at phases before clinical trials. The hPSC-based disease models can also narrow the gap between traditional animal models and clinical trials. One major challenge is the differentiation process of hPSCs into cells of the relevant tissue. The recent study of our laboratory shows that the liver cell-deried acellular matrix (ACM) promotes the hepatic commitment of hESCs. To create chemically defined, xeno-free and feeder-free culture matrices for the differentiation of the hESCs into hepatocyte-like cells (HLCs), the ECM components of the ACM were characterized. The results suggest that the ACM contains fibronectin, laminins. After the characterization, the object was to identify which of the ECM proteins are essential and effective in the differentiation. A three-step differentiation protocol with differenent ECM protein solutions was used to produce HLCs. The hESCs were first induced into definitive endoderm (DE) cells. The DE cells were committed to the bipotential hepatic progenitors positive for HNF4α and AFP. Finally the progenitors were differentiated into HLCs. The mRNA expression of albumin, CK8, CK18, AAT, and BCRP was increased in HLCs. All the derived HLCs were albumin positive. The hESCderived HLCs showed hepatic morphology, cytoplasmic vacuole characteristics, and functional albumin secretion. The chemically defined matrices showed a supportive role in the differentiation of the hESCs into HLCs. This study establishes an efficient, chemically defined, xeno-free system to produce HLCs as a cell source for pharmaceutical and developmental studies.
-
(2013)Laminins are a family of heterotrimeric glycoproteins found mainly in basement membranes. They interact with numerous other extracellular matrix components and cell surface receptors, including integrins and α-dystroglycan. Laminins play roles in myriad of functions including tissue morphogenesis, organogenesis, maintenance of tissue integrity and compartmentalization. In central nervous system laminins are involved in every major developmental stage from neural tube closure to synaptogenesis. Laminin expression in central nervous system decreases after maturation but has been found inducible by injury after trauma or disease. Since laminins are known to promote neurite outgrowth and neuronal survival, this has been proposed as a regenerative response to injury. Although the effects of endogenous laminin are clearly inadequate for repair, laminin based compounds could be powerful therapeutic agents. In previous in vivo studies KDI-tripeptide, a neurite outgrowth promoting fragment from γ1-laminin, has proved effective neuroprotective and regeneration promoting compound. Encouraged by these results I set out to test whether KDI would rescue midbrain dopaminergic neurons in unilateral 6-hydroxydopamine-induced rat model of Parkinson's disease. KDI (1-30µg) was injected to the striatum six hours prior to 6-hydroxydopamine. The severity of the lesion was then evaluated by measuring D-amphetamine induced rotation 2, 4 and 6 weeks postlesion and by assessing the number of neurons in substantia nigra pars compacta and optical density of striatum after tyrosine hydroxylase immunostaining at week seven. The only effective KDI dose studied was 3 µg. Compared to control it decreased Damphetamine induced rotational behaviour significantly at week four. KDI, however, failed to save tyrosine hydroxylase positive dopaminergic neurons in substantia nigra pars compacta or their axons in striatum. KDI might be usable in treating Parkinson's disease but it's mode of action doesn't appear to rely on protecting dopaminergic neurons or promoting the branching of their axons. KDI is known to inhibit ionotropic glutamate receptors and could therefore improve motor function by opposing striatal denervation induced overactivity of glutamatergic subthalamic nucleus neurons.
-
(2023)Extracellular matrix components such as laminins have important roles in supporting the mammary gland epithelium and guiding its development and homeostasis. Adhesion to laminin alpha-5 subunit (Lama5), notably secreted by the hormone receptor positive luminal epithelial cells, promotes luminal epithelial differentiation and cellular identity, as well as controls mammary progenitor activity, contributing to physiological growth of the mammary epithelium. Lama5 loss in luminal mammary epithelial cells results in abnormal epithelial differentiation, aberrated ductal development, and diminished mammary epithelial growth in mice. Breast cancer is one of the most common forms of cancer, and the most common subtypes are hormone receptor positive luminal breast cancers. While expression of other epithelial laminin alpha subunits is often lost, Lama5 is commonly overexpressed in human breast cancer cells, notably in luminal cancer subtypes, as opposed to basal-like and other cancers subtypes. However, the role of Lama5 in mammary tumor growth and identity has not been experimentally studied in neither mice nor humans. In this thesis, the role of Lama5 in the growth and identity of mammary tumors was studied using both in vivo mouse and in vitro human approaches. Lama5 deletion in luminal epithelial cells of tumor-bearing mice was shown to result in five-fold decrease in the amount of tumorous growth in mouse mammary glands in vivo. In vitro LAMA5 downregulation of MCF-7 luminal human breast cancer cells was shown to lower the proliferation rate and increase the doubling time in 2D culture, decrease their mammosphere forming capacity, as well as decrease total growth in 3D culture, while no effects were observed in triple-negative basal-like MDA-MB-231 human breast cancer cells with LAMA5 downregulation. Additionally, downregulation of LAMA5 was shown to promote the expression of basal-like breast cancer and EMT markers vimentin and fibronectin in luminal MCF-7 cells, while the expression of luminal identity markers was not altered. No changes in the expression of luminal or basal cytokeratin markers CK8 and CK14 were seen in mammary tumors in vivo on mice with luminal laminin alpha-5 deletion. This thesis provides the first set of experimental evidence of the role of laminin alpha-5 as a factor promoting mammary tumor growth in both mice and human cells, especially in hormone receptor positive luminal cancer types. Lama5 contribution to tumor identity, and the exact mechanisms require further studies.
Now showing items 1-5 of 5