| 1. |
- Brolen, Gabriella, et al.
(författare)
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Hepatocyte-like cells derived from human embryonic stem cells specifically via definitive endoderm and a progenitor stage
- 2010
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 145:3, s. 284-294
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells offer a potential unlimited supply for functional hepatocytes, since they can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing various hepatic markers. These cells could be used in various applications such as studies of drug metabolism and hepatotoxicity, which however, would require a significant expression of drug metabolizing enzymes. To derive these cells we use a stepwise differentiation protocol where growth- and maturation factors are added. The first phase involves the formation of definitive endoderm. Next, these cells are treated with factors known to promote the induction and proliferation towards hepatic progenitor cell types. In the last phase the cells are terminally differentiated and maturated into functional hepatocyte-like cells. The cultures were characterized by analysis of endodermal or hepatic markers and compared to cultures derived without induction via definitive endoderm. Hepatic functions such as urea secretion, glycogen storage, indocyanine green uptake and secretion, and cytochrome P450-expression and activity were evaluated. The DE-Hep showed a hepatocyte morphology with sub-organized cells and exhibited many liver-functions including transporter activity and capacity to metabolize drugs specific for important cytochrome P450 sub-families. This represents an importantstep in differentiation of hESC into functional hepatocytes. (C) 2009 Elsevier B.V. All rights reserved.
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| 2. |
- Sivertsson, Louise, et al.
(författare)
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Hepatic differentiation and maturation of human embryonic stem cells cultured in a perfused three-dimensional bioreactor
- 2013
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Ingår i: Stem Cells and Development. - : Mary Ann Liebert. - 1547-3287 .- 1557-8534. ; 22:4, s. 581-594
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Tidskriftsartikel (refereegranskat)abstract
- Drug-induced liver injury is a serious and frequently occurring adverse drug reaction in the clinics and is hard to predict during preclinical studies. Today, primary hepatocytes are the most frequently used cell model for drug discovery and prediction of toxicity. However, their use is marred by high donor variability regarding drug metabolism and toxicity, and instable expression levels of liver-specific genes such as cytochromes P450. An in vitro model system based on human embryonic stem cells (hESC), with their unique properties of pluripotency and self-renewal, has potential to provide a stable and unlimited supply of human hepatocytes. Much effort has been made to direct hESC toward the hepatic lineage, mostly using 2-dimensional (2D) cultures. Although the results are encouraging, these cells lack important functionality. Here, we investigate if hepatic differentiation of hESC can be improved by using a 3-dimensional (3D) bioreactor system. Human ESCs were differentiated toward the hepatic lineage using the same cells in either the 3D or 2D system. A global transcriptional analysis identified important differences between the 2 differentiation regimes, and we identified 10 pathways, highly related to liver functions, which were significantly upregulated in cells differentiated in the bioreactor compared to 2D control cultures. The enhanced hepatic differentiation observed in the bioreactor system was also supported by immunocytochemistry. Taken together, our results suggest that hepatic differentiation of hESC is improved when using this 3D bioreactor technology as compared to 2D culture systems.
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| 3. |
- Sivertsson, Louise
(författare)
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Novel human in vitro systems for studies of drug inducted hepatotoxicity
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Drug-induced liver injury (DILI) is a major human health concern, estimated to account for about half of all cases of acute liver failure in the general population. DILI also represents a significant problem in drug discovery, being one of the most common reasons for regulatory actions, including boxed warnings, restricted marketing and withdrawals of marketed drugs. Adverse drug effects in the liver are hard to detect at an early stage during drug development, much owing to the shortcomings of the currently available pre-clinical model systems. The work presented in this thesis aimed to refine and further develop more sensitive, human in vitro models and methods for better prediction of DILI and the underlying mechanisms. Mono-culture of human primary hepatocytes is the closest in vitro model to the human liver, currently considered the golden standard in drug development. However, limitations, such as low availability of qualitative liver tissue and phenotypical instability of these cells in culture, require new sources of functional human hepatic cells. In this thesis, we have shown that high-density culture of the human hepatoma cell line Huh7 induces a spontaneous, hepatic differentiation process, without the need for inducers as is usually the case. A particular increase of CYP3A4 gene- and protein expression and catalytically activity was observed. Moreover, we found that the large increase in CYP3A4 expression seen in the confluent Huh7 cells is mediated by PXR nuclear translocation and increased PXR mediated transcriptional activity, most likely as a result of decreased CDK2 activity and cell cycle arrest. The high constitutive expression of CYP3A4 in the confluent Huh7 cells makes this cell system useful for studies of mechanisms for regulation of PXR and the CYP3A4 gene. The unique characteristics of stem cells make them an attractive large-scale source of hepatic cells for drug development and safety assessment. Using a novel stepwise differentiation protocol we have been able to differentiate human embryonic stem cells (hESC), via definitive endoderm and progenitor stages to hepatocyte-like cells which exhibit many hepatocyte-specific features and functions, including CYP metabolic activities. A dynamic three-dimensional (3D) bioreactor system was shown to prolong and maintain the specific functions of primary hepatocytes, as well as facilitate the hepatic maturation of hESC into hepatocyte-like cells. It has become increasingly evident that inflammatory event plays a significant role in many DILI events. Thus, in vitro systems containing a population of immune competent cells in combination with hepatic cells could be of great significance for studying mechanisms underlying DILI. A co-culture cell model consisting of hepatocytes and monocytes has been developed where the cells were separated by a semipermeable membrane. The hepatotoxic drug troglitazone caused a potentiated and more rapid cytotoxic effect in cells treated in the co-culture compared to the single cultures. Troglitazone treatment also resulted in an increased expression of several stress-related genes in the co-cultures compared to the single cultures. These results suggest a synergistic cytotoxic effect by soluble mediators released by the cells and underscores the importance of incorporating several different hepatic cell typesin order to generate more sensitive in vitro systems and better prediction of DILI.
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