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Starokozhko, V., Vatakuti, S., Schievink, B. H., Merema, M. T., Asplund, A., Synnergren, J., . . . Groothuis, G. M. M. (2017). Maintenance of drug metabolism and transport functions in human precision-cut liver slices during prolonged incubation for 5 days. Archives of Toxicology, 91(5), 2079-2092
Open this publication in new window or tab >>Maintenance of drug metabolism and transport functions in human precision-cut liver slices during prolonged incubation for 5 days
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2017 (English)In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 91, no 5, p. 2079-2092Article in journal (Refereed) Published
Abstract [en]

Human precision-cut liver slices (hPCLS) are a valuable ex vivo model that can be used in acute toxicity studies. However, a rapid decline in metabolic enzyme activity limits their use in studies that require a prolonged xenobiotic exposure. The aim of the study was to extend the viability and function of hPCLS to 5 days of incubation. hPCLS were incubated in two media developed for long-term culture of hepatocytes, RegeneMed(®), and Cellartis(®), and in the standard medium WME. Maintenance of phase I and II metabolism was studied both on gene expression as well as functional level using a mixture of CYP isoform-specific substrates. Albumin synthesis, morphological integrity, and glycogen storage was assessed, and gene expression was studied by transcriptomic analysis using microarrays with a focus on genes involved in drug metabolism, transport and toxicity. The data show that hPCLS retain their viability and functionality during 5 days of incubation in Cellartis(®) medium. Albumin synthesis as well as the activity and gene expression of phase I and II metabolic enzymes did not decline during 120-h incubation in Cellartis(®) medium, with CYP2C9 activity as the only exception. Glycogen storage and morphological integrity were maintained. Moreover, gene expression changes in hPCLS during incubation were limited and mostly related to cytoskeleton remodeling, fibrosis, and moderate oxidative stress. The expression of genes involved in drug transport, which is an important factor in determining the intracellular xenobiotic exposure, was also unchanged. Therefore, we conclude that hPCLS cultured in Cellartis(®) medium are a valuable human ex vivo model for toxicological and pharmacological studies that require prolonged xenobiotic exposure.

Place, publisher, year, edition, pages
Springer, 2017
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Bioinformatics; INF502 Biomarkers
Identifiers
urn:nbn:se:his:diva-13046 (URN)10.1007/s00204-016-1865-x (DOI)000399875300004 ()27717970 (PubMedID)2-s2.0-84991020738 (Scopus ID)
Available from: 2016-10-24 Created: 2016-10-24 Last updated: 2019-11-25Bibliographically approved
Ghosheh, N., Olsson, B., Edsbagge, J., Küppers-Munther, B., Van Giezen, M., Asplund, A., . . . Synnergren, J. (2016). Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines. Stem Cells International, 2016, Article ID 8648356.
Open this publication in new window or tab >>Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines
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2016 (English)In: Stem Cells International, ISSN 1687-9678, Vol. 2016, article id 8648356Article in journal (Refereed) Published
Abstract [en]

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.

Place, publisher, year, edition, pages
Hindawi Publishing Corporation, 2016
National Category
Cell Biology
Research subject
Bioinformatics
Identifiers
urn:nbn:se:his:diva-12033 (URN)10.1155/2016/8648356 (DOI)000373503900001 ()26949401 (PubMedID)2-s2.0-84959330405 (Scopus ID)
Available from: 2016-03-14 Created: 2016-03-14 Last updated: 2019-11-26Bibliographically approved
Asplund, A., Pradip, A., van Giezen, M., Aspegren, A., Choukair, H., Rehnström, M., . . . Küppers-Munther, B. (2016). One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells. Stem Cell Reviews, 12(1), 90-104
Open this publication in new window or tab >>One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells
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2016 (English)In: Stem Cell Reviews, ISSN 1550-8943, E-ISSN 1558-6804, Vol. 12, no 1, p. 90-104Article in journal (Refereed) Published
Abstract [en]

Human hepatocytes display substantial functional inter-individual variation regarding drug metabolizing functions. In order to investigate if this diversity is mirrored in hepatocytes derived from different human pluripotent stem cell (hPSC) lines, we evaluated 25 hPSC lines originating from 24 different donors for hepatic differentiation and functionality. Homogenous hepatocyte cultures could be derived from all hPSC lines using onestandardized differentiation procedure. To the best of our knowledge this is the first report of a standardized hepatic differentiation procedure that is generally applicable across a large panel of hPSC lines without any adaptations to individual lines. Importantly, with regard to functional aspects, such as Cytochrome P450 activities, we observed that hepatocytes derived from different hPSC lines displayed inter-individual variation characteristic for primary hepatocytes obtained from different donors, while these activities were highly reproducible between repeated experiments using the same line. Taken together, these data demonstrate the emerging possibility to compile panels of hPSC-derived hepatocytes of particular phenotypes/genotypes relevant for drug metabolism and toxicity studies. Moreover, these findings are of significance for applications within the regenerative medicine field, since our stringent differentiation procedure allows the derivation of homogenous hepatocyte cultures from multiple donors which is a prerequisite for the realization of future personalized stem cell based therapies.

Place, publisher, year, edition, pages
Springer, 2016
Keywords
Cellular therapy, Hepatocyte differentiation, Human embyronic stem cells, Human induced pluripotent stem cells, Liver, Toxicity
National Category
Cell and Molecular Biology
Research subject
Medical sciences; Bioinformatics
Identifiers
urn:nbn:se:his:diva-11781 (URN)10.1007/s12015-015-9621-9 (DOI)000374582000008 ()26385115 (PubMedID)2-s2.0-84955335024 (Scopus ID)
Available from: 2015-12-31 Created: 2015-12-31 Last updated: 2018-07-31Bibliographically approved
Godoy, P., Schmidt-Heck, W., Natarajan, K., Lucendo-Villarin, B., Szkolnicka, D., Asplund, A., . . . Hengstler, J. G. (2015). Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells. Journal of Hepatology, 63(4), 934-942
Open this publication in new window or tab >>Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells
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2015 (English)In: Journal of Hepatology, ISSN 0168-8278, E-ISSN 1600-0641, Vol. 63, no 4, p. 934-942Article in journal (Refereed) Published
Abstract [en]

BACKGROUND & AIMS: The differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories.

METHODS: Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified.

RESULTS: Gene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The "unwanted" intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC.

CONCLUSIONS: The present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation.

Place, publisher, year, edition, pages
Elsevier, 2015
National Category
Cell Biology
Research subject
Bioinformatics
Identifiers
urn:nbn:se:his:diva-11775 (URN)10.1016/j.jhep.2015.05.013 (DOI)000361729900022 ()26022688 (PubMedID)2-s2.0-84941935050 (Scopus ID)
Available from: 2015-12-21 Created: 2015-12-21 Last updated: 2018-07-31Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-1174-3611

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