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Wegler, C., Ölander, M., Lundquist, P. & Artursson, P. (2020). Global variability analysis of mRNA and protein concentrations across and within human tissues. NAR genomics and bioinformatics, 2(1), Article ID lqz010.
Open this publication in new window or tab >>Global variability analysis of mRNA and protein concentrations across and within human tissues
2020 (English)In: NAR genomics and bioinformatics, ISSN 2631-9268, Vol. 2, no 1, article id lqz010Article in journal (Refereed) Epub ahead of print
Abstract [en]

Genes and proteins show variable expression patterns throughout the human body. However, it is not clear whether relative differences in mRNA concentrations are retained on the protein level. Furthermore, inter-individual protein concentration variability within single tissue types has not been comprehensively explored. Here, we used the Gini index for in-depth concentration variability analysis of publicly available transcriptomics and proteomics data, and of an in-house proteomics dataset of human liver and jejunum from 38 donors. We found that the transfer of concentration variability from mRNA to protein is limited, that established ‘reference genes’ for data normalization vary markedly at the protein level, that protein concentrations cover a wide variability spectrum within single tissue types, and that concentration variability analysis can be a convenient starting point for identifying disease-associated proteins and novel biomarkers. Our results emphasize the importance of considering individual concentration levels, as opposed to population averages, for personalized systems biology analysis.

Keywords
Gini index, human liver, human intestine, protein variability
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-396193 (URN)10.1093/nargab/lqz010 (DOI)
Funder
Swedish Research Council, 5715
Available from: 2019-10-30 Created: 2019-10-30 Last updated: 2019-10-31Bibliographically approved
Ölander, M., Wiśniewski, J. R., Flörkemeier, I., Handin, N., Urdzik, J. & Artursson, P. (2019). A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes. Archives of Toxicology, 93(3), 819-829
Open this publication in new window or tab >>A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes
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2019 (English)In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 93, no 3, p. 819-829Article in journal (Refereed) Published
Abstract [en]

Primary human hepatocytes are used in all facets of liver research, from in vitro studies of xenobiotic disposition and toxicity to the clinical management of liver failure. Unfortunately, cellular stress during isolation and cryopreservation causes a highly unpredictable loss of the ability to attach and form cell-matrix and cell-cell interactions. Reasoning that this problem could be mitigated at the post-thawing stage, we applied label-free quantitative global proteomics to analyze differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, ultimately leading to apoptosis activation. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately post-thawing, which suggested the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. Brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored attachment ability and promoted a differentiated morphology, as well as formation of 3D spheroids. Further, Z-VAD-FMK treatment restored metabolic and transport functions, with maintained sensitivity to hepatotoxic insults. Altogether, this study shows that differentiation and function of suboptimal human hepatocytes can be restored after cryopreservation, thus markedly increasing the availability of these precious cells.

Keywords
Apoptosis, Cellular stress, Cryopreservation, Hepatotoxicity, Human hepatocytes, Proteomics
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-372723 (URN)10.1007/s00204-018-2375-9 (DOI)000463730100019 ()30560367 (PubMedID)
Funder
Swedish Research Council, 2822Swedish Research Council, 01951
Available from: 2019-01-08 Created: 2019-01-08 Last updated: 2019-04-30Bibliographically approved
Ölander, M., Handin, N. & Artursson, P. (2019). Image-based quantification of cell debris as a measure of apoptosis. Analytical Chemistry, 91(9), 5548-5552
Open this publication in new window or tab >>Image-based quantification of cell debris as a measure of apoptosis
2019 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 9, p. 5548-5552Article in journal (Refereed) Published
Abstract [en]

Apoptosis is a controlled form of cell death that can be induced by various diseases and exogenous toxicants. Common apoptosis detection methods rely on fluorescent markers, which necessitates the use of costly reagents and time-consuming labeling procedures. Label-free methods avoid these problems, but often require specialized instruments instead. Here, we utilize apoptotic cell disintegration to develop a novel label-free detection method based on the quantification of subcellular debris particles in bright-field microscopy images. Debris counts show strong correlations with fluorescence-based annexin V staining, and can be used to study concentration-dependent and temporal apoptosis activation. The method is rapid, low-cost, and easy to apply, as the only experimental step comprises bright-field imaging of culture media samples, followed by automated image processing. The late-stage nature of the debris measurement means that the method can complement other, established apoptosis assays, and its accessibility will allow a wider community of researchers to study apoptotic cell death.

National Category
Pharmaceutical Sciences Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-382403 (URN)10.1021/acs.analchem.9b01243 (DOI)000467642100015 ()31001971 (PubMedID)
Funder
Swedish Research Council, 2822Swedish Research Council, 01951
Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-06-19Bibliographically approved
Ölander, M. (2019). Proteomic and Functional Analysis of In Vitro Systems for Studies of Drug Disposition in the Human Small Intestine and Liver. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Proteomic and Functional Analysis of In Vitro Systems for Studies of Drug Disposition in the Human Small Intestine and Liver
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

To reach the bloodstream, an orally administered drug must be absorbed through the small intestine and avoid extensive clearance in the liver. Estimating these parameters in vitro is therefore important in drug discovery and development. This can be achieved with cellular models that simulate human organ function, such as Caco-2 cells and primary hepatocytes. No model fits every scenario, however, and this thesis aimed at using proteomic and functional analysis to better understand and increase the applicability of in vitro models based on Caco-2 cells and human hepatocytes.

First, the proteome of filter-grown Caco-2 cells was analyzed. This included near-complete coverage of enterocyte-related proteins, and over 300 ADME proteins. Further, by scaling uptake transport kinetics from Caco-2 cells to human jejunum, the importance of considering in vitro­-in vivo expression differences to correctly interpret in vitro transport studies was demonstrated.

Focus was then turned to hepatocytes, where proteomics was used as a basis for the successful development of an apoptosis inhibition protocol for restoration of attachment properties and functionality in suboptimal batches of cryopreserved human hepatocytes. As a spin-off project, image-based quantification of cell debris was developed into a novel apoptosis detection method.

Next, the in vivo heterogeneity of human hepatocytes was explored in an in vitro setting, where it was observed that human hepatocyte batches contain a wide range of cell sizes. By separating the cells into different size fractions, it was found that hepatocyte size corresponds to the microarchitectural zone of origin in the liver. Size separation can thus be used to study zonated liver functions in vitro.

Finally, the proteomes of the major types of non-parenchymal liver cells were analyzed, i.e. liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells. The different cell types all had distinctly different proteomes, and the expression of certain important ADME proteins indicated that non-parenchymal cells participate in drug disposition.

In conclusion, this thesis has improved the phenotypic understanding and extended the applicability of Caco-2 cells and primary human hepatocytes, two of the most important in vitro models for studies of small intestinal and hepatic drug disposition.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 59
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 272
Keywords
proteomics, drug disposition, ADMET, drug transport, drug metabolism, hepatotoxicity, small intestine, liver, caco-2, human hepatocytes, cryopreservation, apoptosis, liver zonation, non-parenchymal cells
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-382406 (URN)978-91-513-0668-1 (ISBN)
Public defence
2019-06-14, Room B41, Biomedical center, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-05-21 Created: 2019-04-24 Last updated: 2019-06-18
Ölander, M., Wisniewski, J., Norén, A. & Artursson, P. (2016). To attach or not to attach: factors behind variable adhesion properties of cryopreserved human hepatocytes. Paper presented at 20th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX), OCT 18-22, 2015, Orlando, FL. Drug metabolism reviews (Softcover ed.), 48, 103-103
Open this publication in new window or tab >>To attach or not to attach: factors behind variable adhesion properties of cryopreserved human hepatocytes
2016 (English)In: Drug metabolism reviews (Softcover ed.), ISSN 0360-2532, E-ISSN 1097-9883, Vol. 48, p. 103-103Article in journal, Meeting abstract (Other academic) Published
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-303432 (URN)000380744900213 ()
Conference
20th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX), OCT 18-22, 2015, Orlando, FL
Note

Supplement: 1, Meeting Abstract: P159

Available from: 2016-11-21 Created: 2016-09-19 Last updated: 2018-01-13Bibliographically approved
Karlgren, M., Vildhede, A., Ölander, M., Wisniewski, J., Norén, A. & Artursson, P. (2015). Global Membrane Protein Analysis Of The Human Liver: Application In Predictions Of Atorvastatin Uptake Clearance. Paper presented at 19th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX) / 29th Meeting of the Japanese-Society-for-the-Study-of-Xenobiotics (JSSX), OCT 19-23, 2014, San Francisco, CA. Drug metabolism reviews (Softcover ed.), 47, 245-246
Open this publication in new window or tab >>Global Membrane Protein Analysis Of The Human Liver: Application In Predictions Of Atorvastatin Uptake Clearance
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2015 (English)In: Drug metabolism reviews (Softcover ed.), ISSN 0360-2532, E-ISSN 1097-9883, Vol. 47, p. 245-246Article in journal, Meeting abstract (Other academic) Published
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-279534 (URN)000365611800486 ()
Conference
19th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX) / 29th Meeting of the Japanese-Society-for-the-Study-of-Xenobiotics (JSSX), OCT 19-23, 2014, San Francisco, CA
Available from: 2016-03-02 Created: 2016-03-02 Last updated: 2018-01-10Bibliographically approved
Wegler, C., Ölander, M., Wisniewski, J. R., Lundquist, P., Zettl, K., Åsberg, A., . . . Artursson, P. Global expression variability of proteins across and within human tissues.
Open this publication in new window or tab >>Global expression variability of proteins across and within human tissues
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(English)In: Article in journal (Other academic) Submitted
Place, publisher, year, edition, pages
Uppsala:
Keywords
Expression variability, Human liver, Human jejunum, Proteomics, Transcriptomics, Reference genes
National Category
Cell and Molecular Biology
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-389738 (URN)
Available from: 2019-07-23 Created: 2019-07-23 Last updated: 2019-07-26
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-4502-8184

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