| 1. |
- Asplund, Annika, 1979-, et al.
(author)
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One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells
- 2016
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In: Stem Cell Reviews and Reports. - : Springer Science and Business Media LLC. - 1550-8943 .- 1558-6804. ; 12:1, s. 90-104
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Journal article (peer-reviewed)abstract
- 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 one standardized 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.
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| 2. |
- Ghosheh, Nidal, et al.
(author)
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Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue
- 2017
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In: Physiological Genomics. - : American Physiological Society. - 1094-8341 .- 1531-2267. ; 49:8, s. 430-446
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Journal article (peer-reviewed)abstract
- Hepatocytes derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm, hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins that differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, we identified a module of genes that were expressed at higher levels in the liver tissue samples than in the hPSC-HEP. Considering that hub proteins and modules generally are essential and have important roles in the protein-protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bring this technology to a wider use.
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| 3. |
- Ghosheh, Nidal, et al.
(author)
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Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines
- 2016
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In: Stem Cells International. - : Hindawi Limited. - 1687-966X .- 1687-9678.
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Journal article (peer-reviewed)abstract
- 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.
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| 4. |
- Ghosheh, Nidal, 1975-, et al.
(author)
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Human Pluripotent Stem Cell-Derived Hepatocytes Show Higher Transcriptional Correlation with Adult Liver Tissue than with Fetal Liver Tissue
- 2020
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In: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 5:10, s. 4816-4827
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Journal article (peer-reviewed)abstract
- Human pluripotent stem cell-derived hepatocytes (hPSC-HEP) display many properties of mature hepatocytes, including expression of important genes of the drug metabolizing machinery, glycogen storage, and production of multiple serum proteins. To this date, hPSC-HEP do not, however, fully recapitulate the complete functionality of in vivo mature hepatocytes. In this study, we applied versatile bioinformatic algorithms, including functional annotation and pathway enrichment analyses, transcription factor binding-site enrichment, and similarity and correlation analyses, to datasets collected from different stages during hPSC-HEP differentiation and compared these to developmental stages and tissues from fetal and adult human liver. Our results demonstrate a high level of similarity between the in vitro differentiation of hPSC-HEP and in vivo hepatogenesis. Importantly, the transcriptional correlation of hPSC-HEP with adult liver (AL) tissues was higher than with fetal liver (FL) tissues (0.83 and 0.70, respectively). Functional data revealed mature features of hPSC-HEP including cytochrome P450 enzymes activities and albumin secretion. Moreover, hPSC-HEP showed expression of many genes involved in drug absorption, distribution, metabolism, and excretion. Despite the high similarities observed, we identified differences of specific pathways and regulatory players by analyzing the gene expression between hPSC-HEP and AL. These findings will aid future intervention and improvement of in vitro hepatocyte differentiation protocol in order to generate hepatocytes displaying the complete functionality of mature hepatocytes. Finally, on the transcriptional level, our results show stronger correlation and higher similarity of hPSC-HEP to AL than to FL. In addition, potential targets for further functional improvement of hPSC-HEP were also identified.
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| 5. |
- Holmgren, Gustav, 1983-, et al.
(author)
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Expression Profiling of Human Pluripotent Stem Cell-Derived Cardiomyocytes Exposed to Doxorubicin-Integration and Visualization of Multi-Omics Data
- 2018
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In: Toxicological Sciences. - : Oxford University Press (OUP). - 1096-6080 .- 1096-0929. ; 163:1, s. 182-195
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Journal article (peer-reviewed)abstract
- Anthracyclines, such as doxorubicin, are highly efficient chemotherapeutic agents against a variety of cancers. However, anthracyclines are also among the most cardiotoxic therapeutic drugs presently on the market. Chemotherapeutic-induced cardiomyopathy is one of the leading causes of disease and mortality in cancer survivors. The exact mechanisms responsible for doxorubicin-induced cardiomyopathy are not completely known, but the fact that the cardiotoxicity is dose-dependent and that there is a variation in time-to-onset of toxicity, and gender- and age differences suggests that several mechanisms may be involved. In this study, we investigated doxorubicin-induced cardiotoxicity in human pluripotent stem cell-derived cardiomyocytes using proteomics. In addition, different sources of omics data (protein, mRNA, and microRNA) from the same experimental setup were further combined and analyzed using newly developed methods to identify differential expression in data of various origin and types. Subsequently, the results were integrated in order to generate a combined visualization of the findings. In our experimental model system, we exposed cardiomyocytes derived from human pluripotent stem cells to doxorubicin for up to 2 days, followed by a wash-out period of additionally 12 days. Besides an effect on the cell morphology and cardiomyocyte functionality, the data show a strong effect of doxorubicin on all molecular levels investigated. Differential expression patterns that show a linkage between the proteome, transcriptome, and the regulatory microRNA network, were identified. These findings help to increase the understanding of the mechanisms behind anthracycline-induced cardiotoxicity and suggest putative biomarkers for this condition.
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| 6. |
- Holmgren, Gustav, 1983-, et al.
(author)
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Identification of novel biomarkers for doxorubicin-induced toxicity in human cardiomyocytes derived from pluripotent stem cells
- 2015
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In: Toxicology. - : Elsevier. - 0300-483X .- 1879-3185. ; 328, s. 102-111
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Journal article (peer-reviewed)abstract
- Doxorubicin is a chemotherapeutic agent indicated for the treatment of a variety of cancer types, including leukaemia, lymphomas, and many solid tumours. The use of doxorubicin is, however, associated with severe cardiotoxicity, often resulting in early discontinuation of the treatment. Importantly, the toxic symptoms can occur several years after the termination of the doxorubicin administration. In this study, the toxic effects of doxorubicin exposure have been investigated in cardiomyocytes derived from human embryonic stem cells (hESC). The cells were exposed to different concentrations of doxorubicin for up to 2 days, followed by a 12 day recovery period. Notably, the cell morphology was altered during drug treatment and the cells showed a reduced contractile ability, most prominent at the highest concentration of doxorubicin at the later time points. A general cytotoxic response measured as Lactate dehydrogenase leakage was observed after 2 days' exposure compared to the vehicle control, but this response was absent during the recovery period. A similar dose-dependant pattern was observed for the release of cardiac specific troponin T (cTnT) after 1 day and 2 days of treatment with doxorubicin. Global transcriptional profiles in the cells revealed clusters of genes that were differentially expressed during doxorubicin exposure, a pattern that in some cases was sustained even throughout the recovery period, suggesting that these genes could be used as sensitive biomarkers for doxorubicin-induced toxicity in human cardiomyocytes. The results from this study show that cTnT release can be used as a measurement of acute cardiotoxicity due to doxorubicin. However, for the late onset of doxorubicin-induced cardiomyopathy, cTnT release might not be the most optimal biomarker. As an alternative, some of the genes that we identified as differentially expressed after doxorubicin exposure could serve as more relevant biomarkers, and may also help to explain the cellular mechanisms behind the late onset apoptosis associated with doxorubicin-induced cardiomyopathy.
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| 7. |
- Holmgren, Gustav, et al.
(author)
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Long-Term Chronic Toxicity Testing Using Human Pluripotent Stem Cell-Derived Hepatocytes
- 2014
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In: Drug Metabolism and Disposition. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0090-9556 .- 1521-009X. ; 42:9, s. 1401-1406
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Journal article (peer-reviewed)abstract
- Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new models for in vitro drug testing of, for example, toxicity and pharmacological effects. Late-stage attrition in the pharmaceutical industry is to a large extent caused by selection of drug candidates using nonpredictive preclinical models that are not clinically relevant. The current hepatic in vivo and in vitro models show clear limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to the best of our knowledge, the first report of a long-term toxicity study using hPSC-derived hepatocytes, and the observations support further development and validation of hPSC-based toxicity models for evaluating novel drugs, chemicals, and cosmetics.
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| 8. |
- Holmgren, Gustav, 1983-, et al.
(author)
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MicroRNAs as potential biomarkers for doxorubicin-induced cardiotoxicity
- 2016
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In: Toxicology in Vitro. - : Elsevier BV. - 0887-2333 .- 1879-3177. ; 34, s. 26-34
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Journal article (peer-reviewed)abstract
- Anthracyclines, such as doxorubicin, are well-established, highly efficient anti-neoplastic drugs used for treatment of a variety of cancers, including solid tumors, leukemia, lymphomas, and breast cancer. The successful use of doxorubicin has, however, been hampered by severe cardiotoxic side-effects. In order to prevent or reverse negative side-effects of doxorubicin, it is important to find early biomarkers of heart injury and drug-induced cardiotoxicity. The high stability under extreme conditions, presence in various body fluids, and tissue specificity, makes,microRNAs very suitable as clinical.biomarkers. The present study aimed towards evaluating the early and late effects of doxorubicin on the microRNA expression in cardiomyocytes derived from human pluripotent stem cells. We report on several microRNAs, including miR-34a, miR-34b, miR-187, miR-199a, miR-199b, miR-146a, miR-15b, miR-130a, miR-214, and miR-424, that are differentially expressed upon, and after, treatment with doxorubicin. Investigation of the biological relevance of the identified microRNAs revealed connections to cardiomyocyte function and cardiotoxicity, thus supporting the findings of these microRNAs as potential biomarkers for drug-induced cardiotoxicity.
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| 9. |
- Johansson, Markus, et al.
(author)
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Cardiac hypertrophy in a dish: a human stem cell based mode
- 2020
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In: Biology Open. - : The Company of Biologists. - 2046-6390. ; 9:9
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Journal article (peer-reviewed)abstract
- Cardiac hypertrophy is an important and independent risk factor for the development of heart failure. To better understand the mechanisms and regulatory pathways involved in cardiac hypertrophy, there is a need for improved in vitro models. In this study, we investigated how hypertrophic stimulation affected human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). The cells were stimulated with endothelin-1 (ET-1) for 8, 24, 48, 72, or 96 h. Parameters including cell size, ANP-, proBNP-, and lactate concentration were analyzed. Moreover, transcriptional profiling using RNA-sequencing was performed to identify differentially expressed genes following ET-1 stimulation. The results show that the CMs increase in size by approximately 13% when exposed to ET-1 in parallel to increases in ANP and proBNP protein and mRNA levels. Furthermore, the lactate concentration in the media was increased indicating that the CMs consume more glucose, a hallmark of cardiac hypertrophy. Using RNA-seq, a hypertrophic gene expression pattern was also observed in the stimulated CMs. Taken together, these results show that hiPSC-derived CMs stimulated with ET-1 display a hypertrophic response. The results from this study also provide new molecular insights about the underlying mechanisms of cardiac hypertrophy and may help accelerate the development of new drugs against this condition.
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| 10. |
- Johansson, Markus, et al.
(author)
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Data Mining Identifies CCN2 and THBS1 as Biomarker Candidates for Cardiac Hypertrophy
- 2022
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In: Life-Basel. - : MDPI AG. ; 12:5
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Journal article (peer-reviewed)abstract
- Cardiac hypertrophy is a condition that may contribute to the development of heart failure. In this study, we compare the gene-expression patterns of our in vitro stem-cell-based cardiac hypertrophy model with the gene expression of biopsies collected from hypertrophic human hearts. Twenty-five differentially expressed genes (DEGs) from both groups were identified and the expression of selected corresponding secreted proteins were validated using ELISA and Western blot. Several biomarkers, including CCN2, THBS1, NPPA, and NPPB, were identified, which showed significant overexpressions in the hypertrophic samples in both the cardiac biopsies and in the endothelin-1-treated cells, both at gene and protein levels. The protein-interaction network analysis revealed CCN2 as a central node among the 25 overlapping DEGs, suggesting that this gene might play an important role in the development of cardiac hypertrophy. GO-enrichment analysis of the 25 DEGs revealed many biological processes associated with cardiac function and the development of cardiac hypertrophy. In conclusion, we identified important similarities between ET-1-stimulated human-stem-cell-derived cardiomyocytes and human hypertrophic cardiac tissue. Novel putative cardiac hypertrophy biomarkers were identified and validated on the protein level, lending support for further investigations to assess their potential for future clinical applications.
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