Browsing by Subject "differentiation"

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.

Bipotential gonads are precursor structures for testes and ovaries. Steroidogenic factor-1 (SF1) is one of the most important transcription factors in an embryo needed for the development and maintenance of bipotential gonads. If SF1 is not expressed, bipotential gonads fail to develop, and genitalia and kidneys are not formed. Later, SF1 expression persists high in testes, where it supports Sertoli and Leydig cell formation and development. If SF1 is not expressed enough in males, the bipotential gonads differentiate into ovaries. The factors activating and regulating SF1 are not currently fully known. By getting more knowledge of how SF1 is controlled, regulatory mechanisms behind normal fetal development of gonads and disorders of sex development (DSD) can be understood better. The aims of this thesis were to study whether growth factors, that naturally regulate differentiation of developing gonads, promote differentiation of human induced pluripotent stem cells (hiPSC) into Sertoli-like cells (SLCs) and whether SF1 expression is induced by the addition of these growth factors. For conducting the study, we used hiPSCs, which have an SF1 activation domain cassette previously introduced to the cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) method. SF1 could be activated by adding doxycycline (DOX) and trimethoprim (TMP). These hiPSCs were differentiated into intermediate mesoderm (IM) on the first four days according to the protocol published earlier by the group. After this, the differentiation to SLCs was guided by adding growth factors to the culture medium. Basic fibroblast growth factor (bFGF), fibroblast growth factor 9 (FGF9) and prostaglandin-2 (PGD2) were tested separately and in a combined cocktail also including follicle stimulating (FSH) and glial cell-derived neurotrophic factor (GDNF). In a control condition, cells were differentiated without additional growth factors. In all tested conditions, cells first differentiated into IM were further differentiated either in the presence or absence of DOX and TMP for 8 days. The differentiation medias were changed to the cells every day and lysis samples for quantitative PCR (qRT-PCR) were taken every other day. The relative gene expression levels of bipotential gonad, testis and steroidogenic gene markers from each condition were monitored with qRT-PCR and compared to the levels of the undifferentiated hiPSCs. Immunocytochemistry was performed to see the changes in protein production. Against our hypothesis and the previous studies by others, none of the tested growth factors induced the cells to differentiate into SLCs. However, SF1 expression was triggered by chemical induction with DOX and TMP. Also, the expression levels of bipotential gonadal and testicular gene markers increased in control conditions with/without chemical induction. PGD2 conditions were the only ones to resemble the gene expression and morphology of control conditions while the others differed. These results indicated that the addition of bFGF, FGF9, FSH and GDNF did not improve the differentiation of iPSCs into SLCs and in fact, bFGF and FGF9 hindered their differentiation into SLCs. As a future perspective the optimal concentrations for each growth factor and the duration of growth factor supplementation ought to be tested to refine the protocol.