Browsing by Subject "hepatosyytti"
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(2012)Hepatotoxicity is an undesired feature of many drugs and is one of the main reasons for attrition during the drug development process. Although an in vitro model can never totally correspond to or replace a whole organism, a reliable in vitro model for liver toxicity screening would help to detect liver toxicity earlier in the development process. Effective and early in vitro screening would reduce the need of animal subjects and clinical trials and thus would be both ethically more acceptable and more cost-effective. Currently mostly used models for liver metabolism and toxicity studies are primary hepatocytes, hepatic cell lines and animal models. However, these models have many drawbacks and are not considered reliable. Human embryonic stem cells (hESCs) are pluripotent cells that can be differentiated into many specialized cell types including hepatocytes. They are also self-renewable and thus represent an unlimited and promising source of hepatocytes to be used as a tool in in vitro liver toxicity testing of drug candidates. The aim of this study was to produce hepatocytes from hESCs via multiple steps following the in vivo pathway of developing hepatocytes: first hESCs were differentiated into definitive endoderm cells, after which they were differentiated into hepatic progenitor cells. Finally, hepatocyte-like cells (HLCs) were induced from the progenitor cells. Our specific interest was the use of hepatic cell derived acellular matrix as a differentiation basis for hepatic progenitors and hepatocytes. We also studied the effect of Matrigel overlay on the hepatic differentiation. Differentiation method without the Matrigel overlay was promising. HLCs showed correct hepatocyte-like morphology and expressed hepatocyte markers such as albumin, α-antitrypsin, CYP3A4 and HNF4α both on mRNA and protein level shown by qPCR and flow cytometry and immunofluorescence staining, respectively. Accordingly, the expression of stem cells marker SSEA-3 showed a tendency to decrease as the differentiation proceeded. HLCs also functionally resembled hepatocytes shown by albumin production. However, we could not detect other hepatocyte functions such as urea production or CYP activity. With Matrigel overlay, the hepatocyte-like morphology of the cells was lost, no albumin production was shown and the expression of several hepatocyte markers was lower than in the experiment done without the Matrigel overlay. Thus, Matrigel overlay was shown to be unbeneficial for hepatocyte differentiation. In conclusion, we showed that differentiation of hESCs on the acellular matrix with specific growth factors and without the Matrigel overlay seems promising as a method to produce HLCs. This preliminary study serves as a basis for future studies, in which the differentiation method should still be further studied and developed to yield functional HLCs of uniform quality.
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(2012)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.
Now showing items 1-2 of 2