Browsing by Subject "erilaistaminen"

Pharmaceutical companies are currently facing increasing developmental costs, and at the same time, less new compounds are being brought to the market. In vitro -metabolism studies and toxicity assessment of new drug candidates are crucial, as early as possible, to prevent their withdrawal in later development phases. Used study systems are, however, limited and new improved technologies are being investigated. Notable, drug induced liver toxicity and alterations in the liver function are frequent reasons for the drug removals from the development. Human embryonic stem cell (hESC) is one of the most powerful cell types known. hESCs have not only the possibility to divide indefinitely but these cells have also the ability to differentiate to all mature cell types of the human body, such as hepatocytes. This makes them potentially very valuable for pharmaceutical development, in order to create a functional in vitro -model, mimicking the liver tissue. In the literature part, the three dimensional (3D) -hepatic differentiation of mouse and human ESCs in vitro, are discussed. Traditional 2D-culture systems do not adequately mimic the microenvironment of three dimensionally organized native tissue. In 2D-cultures cells grow as a monolayer, when the cell morphology is flattened leading to poor cell-cell and cell-matrix contacts and preventing from the tissue formation. In 3D-culture systems, cells are able to form tissue-like cell integrations, spheroids, and thus, remain their functionality and viability significantly longer. Hydrogels are commonly used biomaterials in 3D-cell cultivation and well known in various areas of tissue engineering for their nano scale porosity and ability to surround cells in 3D-polymer network. In addition, they are capable to absorb large volumes of water and functionalized, in various ways, to improve the required biological or mechanical properties. In the experimental part, the main purpose was to differentiate human hepatic progenitor cells to mature hepatocyte-like cells in three dimensional (3D) -biomaterials. Overall, four different hydrogels (cellulose nanofiber (CNF) hydrogel, HydroMatrixTM, ExtracelTM and PuraMatrixTM) were used as 3D-cell culture scaffolds. Several hepatic cell functions (albumin and urea production and cytochrome P450 (CYP) 3A4 activity) were measured in 2D- and 3D-cultures and compared with the human hepatic carcinoma cells, HepG2, which are often used in drug development. Differentiated hepatocyte-like cells did not show CYP3A4 activity and they produced less albumin and urea compared with HepG2 cells. However, working with hESCs is very demanding and the research in this area is only in the beginning. Therefore, the poor cell functionality results did not come up as a surprise.

Functional in vitro cultured human hepatocytes are needed in different applications in biomedical research. Treatment for liver diseases is usually liver transplantation, but due to the lack of healthy donors, cell therapy using hepatocytes is considered as a better option. Drug industry will also need representative liver models to test metabolic profiles of drug molecules. Primary human hepatocytes are studied in cell therapy and disease modelling, but they have also drawbacks. In vitro they do not proliferate efficiently, and they are short-lived. In vitro differentiated human pluripotent stem cells (hPSCs) to hepatic fate are an alternative for the primary human hepatocytes. Especially human induced pluripotent stem cells (hiPSCs) are widely studied because they are easily available, and they even make personalized therapy possible without problems with ethical issues related to the human embryonic stem cells (hESCs). Differentiation to hepatic fate includes several steps before mature functional hepatocyte-like cells are formed. Hepatocytes are derived from the definitive endoderm (DE) which is one of the germ layers formed in the gastrulation process. Efficient induction of hPSCs into DE lineage would be a good starting point for generating mature hepatocyte-like cells in further hepatic differentiation. Different protocols to differentiate hPSCs in vitro into DE have been published. In vitro cell culture systems should well represent the environment of the target tissue because signals from the environment guide the differentiation. Three-dimensional (3D) cell culture systems are widely studied, because they better mimic the in vivo microenvironment of cells than two-dimensional (2D) cell culture. The aim of the thesis was to study the efficacy of the 3D differentiation of hiPSCs into DE. Before starting the 3D differentiation, differentiation protocol was optimized and the effect of ROCK inhibitor Y-27632 was investigated. Differentiation medium was supplemented with Y-27632 during the whole 6 days differentiation, because survival of the cells and formation of the spheroids were improved, and gene expression studies of pluripotency markers and several DE markers did not show evident effect of Y-27632 on the gene expression of hiPSCs. The main objective in the studies was also to investigate possible differences between different 3D culture conditions on hiPSCs differentiation into DE. Also, the effect of the spheroid size on differentiation was examined. Two different hydrogels were used as a matrix material in the experiments: basement membrane extract (BME) and nanofibrillar cellulose (NFC) hydrogels. Suspension culture was used as a biomaterial-free 3D culture system. Experiments were performed with three spheroid sizes: 200 cells/spheroid, 500 cells/spheroid and 1000 cells/spheroid. Efficacy of differentiation to DE lineage was estimated by studying protein and mRNA expression of some of the DE markers (HNF3B, SOX17, CXCR4, CER1), pluripotency marker OCT4, mesendoderm marker Brachyury and hepatoblast marker HNF4A in the cells. Spheroids differentiated in suspension and NFC were analysed by flow cytometry to get the number of DE positive live cells and dead cells using CXCR4 and 7-AAD double staining. Besides flow cytometry, protein expression of some of the key markers were studied by immunofluorescent staining and further confocal imaging. Viability of the spheroids in BME hydrogel culture were investigated using live/dead staining followed by confocal imaging. BME hydrogel culture was left out from the further experiments due to the morphology of the spheroids and results from viability and protein expression studies. Spheroids in suspension started DE differentiation faster compared to NFC culture. Suspension and NFC cultures yielded high number of double positive cells in flow cytometry and bright fluorescence of other DE markers was seen in the confocal images. NFC hydrogel proved to be a promising 3D culture system by supporting the differentiation of hiPSCs. Flow cytometry results and gene expression studies propose that four days long 3D differentiation would be efficient to produce sufficient number of DE cells. Smaller spheroids showed higher number of DE positive cells than bigger spheroids on day 2 but gene expression studies showed difference in DE marker expression between size conditions rather in later days in differentiation and it was the opposite. Experiments showed signs of more efficient differentiation of the smaller sized spheroids in the beginning of differentiation. But further studies are needed to verify the obtained results and both draw conclusions about the possible differences between different 3D culture systems and explore the best size of the spheroid for hepatic differentiation. However, results obtained from the studies are useful for designing further experiments.