| 1. |
- Brolén, Gabriella
(author)
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Embryonic Stem Cells: Differentiation into Insulin Producing Cells and Elimination of Damaged Proteins
- 2006
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Doctoral thesis (other academic/artistic)abstract
- This thesis includes two different parts: One focusing on how to induce human embryonic stem cells (hESCs) to differentiate into insulin producing cells by following the normal pancreatic development pathway. These cells have then the potential to be an unlimited source for diabetes regenerative medicine. The second part of the thesis deals with how embryonic stem cells rid themselves of damaged protein. We have discovered that stem cells have a unique mechanism for doing this. Spontaneous differentiation of hESCs (dhESCs) under two-dimensional in vitro growth conditions resulted in differentiation of pancreatic progenitors (Pdx1+/Foxa2+) and endocrine progenitors (Pdx1+/Isl1+) but not into insulin producing cells. However, co-transplantation of dhESCs with pancreatic dorsal bud and epithelium free from mesenchyme, but not with liver or telencephalon, from mouse embryos resulted in derivation of human insulin producing cells clusters with beta cell characteristic. Comparative analysis of these cells with human adult islets demonstrated that the insulin positive cells share important features with normal beta cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key beta cell transcription factors, including Foxa2, Pdx1, Nkx6.1, and Isl1. These results suggest that both the environment of the kidney capsule and instructive signals form embryonic pancreatic bud and epithelium are required to induce dhESCs into insulin producing cells. Unexpectedly, we found that undifferentiated mouse ESCs contain high levels of both carbonyls and AGEs. The damaged proteins, identified as chaperones and proteins of the cytoskeleton are the main targets for protein oxidation in aged tissues. However, our results show that mouse ESCs rid themselves of such damage upon differentiation in vitro and in vivo. This elimination of damaged proteins coincides with a considerably elevated activity of the 20S proteasome. This clear-out of damaged proteins may be a part of a previously unknown rejuvenation process at the protein level and makes it worth considering that the offspring of mammals may initially be free of protein damage because of an early developmental damage elimination rather than by a mechanism that keeps the germ-line cells free of destructive molecules.
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| 2. |
- Brolen, Gabriella, et al.
(author)
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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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In: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 145:3, s. 284-294
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Journal article (peer-reviewed)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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| 3. |
- Brolén, Gabriella, et al.
(author)
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Signals From the Embryonic Mouse Pancreas Induce Differentiation of Human Embryonic Stem Cells Into Insulin-Producing {beta}-Cell-Like Cells.
- 2005
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In: Diabetes. - : American Diabetes Association. - 1939-327X .- 0012-1797. ; 54:10, s. 2867-2874
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Journal article (peer-reviewed)abstract
- The recent success in restoring normoglycemia in type 1 diabetes by islet cell transplantation indicates that cell replacement therapy of this severe disease is achievable. However, the severe lack of donor islets has increased the demand for alternative sources of beta-cells, such as adult and embryonic stem cells. Here, we investigate the potential of human embryonic stem cells (hESCs) to differentiate into beta-cells. Spontaneous differentiation of hESCs under two-dimensional growth conditions resulted in differentiation of Pdx1(+)/Foxa2(+) pancreatic progenitors and Pdx1(+)/Isl1(+) endocrine progenitors but no insulin-producing cells. However, cotransplantation of differentiated hESCs with the dorsal pancreas, but not with the liver or telencephalon, from mouse embryos resulted in differentiation of beta-cell-like cell clusters. Comparative analysis of the basic characteristics of hESC-derived insulin(+) cell clusters with human adult islets demonstrated that the insulin(+) cells share important features with normal beta-cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key beta-cell transcription factors, including Foxa2, Pdx1, and Isl1.
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| 4. |
- Carlsson, Björn, et al.
(author)
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Review article: the emerging role of genetics in precision medicine for patients with non-alcoholic steatohepatitis.
- 2020
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In: Alimentary pharmacology & therapeutics. - : Wiley. - 1365-2036 .- 0269-2813. ; 51:12, s. 1305-1320
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Research review (peer-reviewed)abstract
- Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD) characterised by liver fat accumulation, inflammation and progressive fibrosis. Emerging data indicate that genetic susceptibility increases risks of NAFLD, NASH and NASH-related cirrhosis.To review NASH genetics and discuss the potential for precision medicine approaches to treatment.PubMed search and inclusion of relevant literature.Single-nucleotide polymorphisms in PNPLA3, TM6SF2, GCKR, MBOAT7 and HSD17B13 are clearly associated with NASH development or progression. These genetic variants are common and have moderate-to-large effect sizes for development of NAFLD, NASH and hepatocellular carcinoma (HCC). The genes play roles in lipid remodelling in lipid droplets, hepatic very low-density lipoprotein (VLDL) secretion and de novo lipogenesis. The PNPLA3 I148M variant (rs738409) has large effects, with approximately twofold increased odds of NAFLD and threefold increased odds of NASH and HCC per allele. Obesity interacts with PNPLA3 I148M to elevate liver fat content and increase rates of NASH. Although the isoleucine-to-methionine substitution at amino acid position 148 of the PNPLA3 enzyme inactivates its lipid remodelling activity, the effect of PNPLA3 I148M results from trans-repression of another lipase (ATGL/PNPLA2) by sequestration of a shared cofactor (CGI-58/ABHD5), leading to decreased hepatic lipolysis and VLDL secretion. In homozygous Pnpla3 I148M knock-in rodent models of NAFLD, targeted PNPLA3 mRNA knockdown reduces hepatic steatosis, inflammation and fibrosis.The emerging genetic and molecular understanding of NASH paves the way for novel interventions, including precision medicines that can modulate the activity of specific genes associated with NASH.
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| 5. |
- Delsing, Louise, et al.
(author)
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Barrier properties and transcriptome expression in human iPSC-derived models of the blood-brain barrier
- 2018
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In: Stem Cells. - : AlphaMed Press, Inc.. - 1066-5099 .- 1549-4918. ; 36:12, s. 1816-1827
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Journal article (peer-reviewed)abstract
- Cell-based models of the blood-brain barrier (BBB) are important for increasing the knowledge of BBB formation, degradation and brain exposure of drug substances. Human models are preferred over animal models because of inter-species differences in BBB structure and function. However, access to human primary BBB tissue is limited and has shown degeneration of BBB functions in vitro. Human induced pluripotent stem cells (iPSCs) can be used to generate relevant cell types to model the BBB with human tissue. We generated a human iPSC-derived model of the BBB that includes endothelial cells in co-culture with pericytes, astrocytes and neurons. Evaluation of barrier properties showed that the endothelial cells in our co-culture model have high transendothelial electrical resistance, functional efflux and ability to discriminate between CNS permeable and non-permeable substances. Whole genome expression profiling revealed transcriptional changes that occur in co-culture, including upregulation of tight junction proteins such as claudins and neurotransmitter transporters. Pathway analysis implicated changes in the WNT, TNF and PI3K-Akt pathways upon co-culture. Our data suggests that co-culture of iPSC-derived endothelial cells promotes barrier formation on a functional and transcriptional level. The information about gene expression changes in co-culture can be used to further improve iPSC-derived BBB models through selective pathway manipulation.
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| 6. |
- Doktorova, Tatyana Y., et al.
(author)
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Transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models
- 2013
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In: Carcinogenesis. - : Oxford University Press. - 0143-3334 .- 1460-2180. ; 34:6, s. 1393-1402
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Journal article (peer-reviewed)abstract
- As the conventional approach to assess the potential of a chemical to cause cancer in humans still includes the 2-year rodent carcinogenicity bioassay, development of alternative methodologies is needed. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically stabilized cultures of primary rat hepatocytes, the human hepatoma-derived cell lines HepaRG and HepG2 and human embryonic stem cell-derived hepatocyte-like cells, are examined. For full characterization of the systems, several bioinformatics approaches are employed including gene-based, ConsensusPathDB-based and classification analysis. They provide convincingly similar outcomes, namely that upon exposure to carcinogens, the HepaRG generates a gene classifier (a gene classifier is defined as a selected set of characteristic gene signatures capable of distinguishing GTX, NGTX carcinogens and NC) able to discriminate the GTX carcinogens from the NGTX carcinogens and NC. The other in vitro models also yield cancer-relevant characteristic gene groups for the GTX exposure, but some genes are also deregulated by the NGTX carcinogens and NC. Irrespective of the tested in vitro model, the most uniformly expressed pathways following GTX exposure are the p53 and those that are subsequently induced. The NGTX carcinogens triggered no characteristic cancer-relevant gene profiles in all liver-based in vitro systems. In conclusion, liver-based in vitro models coupled with transcriptomics techniques, especially in the case when the HepaRG cell line is used, represent valuable tools for obtaining insight into the mechanism of action and identification of GTX carcinogens.
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| 7. |
- Granéli, Cecilia, et al.
(author)
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Diabetic Cardiomyopathy Modelling Using Induced Pluripotent Stem Cell Derived Cardiomyocytes : Recent Advances and Emerging Models
- 2019
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In: Stem Cell Reviews. - : Springer. - 1550-8943 .- 1558-6804. ; 15:1, s. 13-22
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Journal article (peer-reviewed)abstract
- The global burden of diabetes has drastically increased over the past decades and in 2017 approximately 4 million deaths were caused by diabetes and cardiovascular complications. Diabetic cardiomyopathy is a common complication of diabetes with early manifestations of diastolic dysfunction and left ventricular hypertrophy with subsequent progression to systolic dysfunction and ultimately heart failure. An in vitro model accurately recapitulating key processes of diabetic cardiomyopathy would provide a useful tool for investigations of underlying disease mechanisms to further our understanding of the disease and thereby potentially advance treatment strategies for patients. With their proliferative capacity and differentiation potential, human induced pluripotent stem cells (iPSCs) represent an appealing cell source for such a model system and cardiomyocytes derived from induced pluripotent stem cells have been used to establish other cardiovascular related disease models. Here we review recently made advances and discuss challenges still to be overcome with regard to diabetic cardiomyopathy models, with a special focus on iPSC-based systems. Recent publications as well as preliminary data presented here demonstrate the feasibility of generating cardiomyocytes with a diabetic phenotype, displaying insulin resistance, impaired calcium handling and hypertrophy. However, capturing the full metabolic- and functional phenotype of the diabetic cardiomyocyte remains to be accomplished.
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| 8. |
- Hernebring, Malin, 1978, et al.
(author)
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Elimination of damaged proteins during differentiation of embryonic stem cells
- 2006
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 103:20, s. 7700-7705
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Journal article (peer-reviewed)abstract
- During mammalian aging, cellular proteins become increasingly damaged: for example, by carbonylation and formation of advanced glycation end products (AGEs). The means to ensure that offspring are born without such damage are unknown. Unexpectedly, we found that undifferentiated mouse ES cells contain high levels of both carbonyls and AGEs. The damaged proteins, identified as chaperones and proteins of the cytoskeleton, are the main targets for protein oxidation in aged tissues. However, the mouse ES cells rid themselves of such damage upon differentiation in vitro. This elimination of damaged proteins coincides with a considerably elevated activity of the 20S proteasome. Moreover, damaged proteins were primarily observed in the inner cell mass of blastocysts, whereas the cells that had embarked on differentiation into the trophectoderm displayed drastically reduced levels of protein damage. Thus, the elimination of protein damage occurs also during normal embryonic development in vivo. This clear-out of damaged proteins may be a part of a previously unknown rejuvenation process at the protein level that occurs at a distinct stage during early embryonic development
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| 9. |
- Lundin, Anders, et al.
(author)
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Human iPS-Derived Astroglia from a Stable Neural Precursor State Show Improved Functionality Compared with Conventional Astrocytic Models
- 2018
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In: Stem Cell Reports. - : Cell Press. - 2213-6711. ; 10:3, s. 1030-1045
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Journal article (peer-reviewed)abstract
- In vivo studies of human brain cellular function face challenging ethical and practical difficulties. Animal models are typically used but display distinct cellular differences. One specific example is astrocytes, recently recognized for contribution to neurological diseases and a link to the genetic risk factor apolipoprotein E (APOE). Current astrocytic in vitro models are questioned for lack of biological characterization. Here, we report human induced pluripotent stem cell (hiPSC)-derived astroglia (NES-Astro) developed under defined conditions through long-term neuroepithelial-like stem (ltNES) cells. We characterized NES-Astro and astrocytic models from primary sources, astrocytoma (CCF-STTG1), and hiPSCs through transcriptomics, proteomics, glutamate uptake, inflammatory competence, calcium signaling response, and APOE secretion. Finally, we assess modulation of astrocyte biology using APOE-annotated compounds, confirming hits of the cholesterol biosynthesis pathway in adult and hiPSC-derived astrocytes. Our data show large diversity among astrocytic models and emphasize a cellular context when studying astrocyte biology.
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| 10. |
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