| 1. |
- Améen, Caroline, 1975, et al.
(författare)
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Human embryonic stem cells: current technologies and emerging industrial applications.
- 2008
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Ingår i: Critical reviews in oncology/hematology. - : Elsevier BV. - 1040-8428. ; 65:1, s. 54-80
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Forskningsöversikt (refereegranskat)abstract
- The efficiency and accuracy of the drug development process is severely restricted by the lack of functional human cell systems. However, the successful derivation of pluripotent human embryonic stem (hES) cell lines in the late 1990s is expected to revolutionize biomedical research in many areas. Due to their growth capacity and unique developmental potential to differentiate into almost any cell type of the human body, hES cells have opened novel avenues both in basic and applied research as well as for therapeutic applications. In this review we describe, from an industrial perspective, the basic science that underlies the hES cell technology and discuss the current and future prospects for hES cells in novel and improved stem cell based applications for drug discovery, toxicity testing as well as regenerative medicine.
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| 2. |
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| 3. |
- Barone, Angela, et al.
(författare)
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Structural Complexity of Non-acid Glycosphingolipids in Human Embryonic Stem Cells Grown under Feeder-free Conditions.
- 2013
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 288:14, s. 10035-50
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Tidskriftsartikel (refereegranskat)abstract
- Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 10(9) cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Le(x) pentaosylceramide, and the Le(y) hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated.
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| 4. |
- Doktorova, Tatyana Y., et al.
(författare)
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Transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models
- 2013
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Ingår i: Carcinogenesis. - : Oxford University Press. - 0143-3334 .- 1460-2180. ; 34:6, s. 1393-1402
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Ghosheh, Nidal, 1975-, et al.
(författare)
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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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Ingår i: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 5:10, s. 4816-4827
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Godoy, Patricio, et al.
(författare)
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Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells
- 2015
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Ingår i: Journal of Hepatology. - : Elsevier. - 0168-8278 .- 1600-0641. ; 63:4, s. 934-942
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Heins, Nico, et al.
(författare)
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Clonal derivation and characterization of human embryonic stem cell lines.
- 2006
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Ingår i: Journal of biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 122:4, s. 511-20
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESC) are isolated as clusters of cells from the inner cell mass of blastocysts and thus should formally be considered as heterogeneous cell populations. Homogenous hESC cultures can be obtained through subcloning. Here, we report the clonal derivation and characterization of two new hESC lines from the parental cell line SA002 and the previously clonally derived cell line AS034.1, respectively. The hESC line SA002 was recently reported to have an abnormal karyotype (trisomy 13), but within this population of cells we observed rare individual cells with an apparent normal karyotype. At a cloning efficiency of 5%, we established 33 subclones from SA002, out of which one had a diploid karyotype and this subline was designated SA002.5. From AS034.1 we established one reclone designated AS034.1.1 at a cloning efficiency of 0.1%. These two novel sublines express cell surface markers indicative of undifferentiated hESC (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), Oct-4, alkaline phosphatase, and they display high telomerase activity. In addition, the cells are pluripotent and form derivatives of all three embryonic germ layers in vitro as well as in vivo. These results, together with the clonal character of SA002.5 and AS034.1.1 make these homogenous cell populations very useful for hESC based applications in drug development and toxicity testing. In addition, the combination of the parental trisomic hESC line SA002 and the diploid subclone SA002.5 provides a unique experimental system to study the molecular mechanisms underlying the pathologies associated with trisomy 13.
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| 8. |
- Mandenius, Carl-Fredrik, et al.
(författare)
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Toward Preclinical Predictive Drug Testing for Metabolism and Hepatotoxicity by Using In Vitro Models Derived from Human Embryonic Stem Cells and Human Cell Lines - A Report on the Vitrocellomics EU-project
- 2011
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Ingår i: ATLA-ALTERNATIVES TO LABORATORY ANIMALS. - : Fund for Replacement of Animals in Medical Experiments; 1999. - 0261-1929 .- 2632-3559. ; 39:2, s. 147-171
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Tidskriftsartikel (refereegranskat)abstract
- Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.
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| 9. |
- Mölne, Johan, 1958, et al.
(författare)
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Blood group ABO antigen expression in human embryonic stem cells and in differentiated hepatocyte- and cardiomyocyte-like cells.
- 2008
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Ingår i: Transplantation. - 1534-6080. ; 86:10, s. 1407-13
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The use of stem cells in regenerative medicine and transplantation may require grafting of cells that will challenge the recipient's immune system. Our knowledge of tissue antigen expression in human embryonic stem cells (hESC) and during their differentiation is limited, especially regarding histo-blood group AB(O)H antigens. METHODS: Nine different hESC lines, and hESC-derived hepatocyte- and cardiomyocyte-like cells, were blood group ABO genotyped and A/B antigen expression was studied by immunohistochemistry. RESULTS: This study reveals, for the first time, that A and B antigens in hESC were expressed according to the ABO genotype and that the antigens had a different cellular/sub-cellular distribution. In addition, several genotype A hESC lines stained positive with one anti-B antibody. Furthermore, studies of hepatocyte- and cardiomyocyte-like cells of different maturation state, originating from a blood group B hESC line, showed that hepatocyte-like cells expressed B antigens whereas cardiomyocyte-like cells were negative. CONCLUSION: Since clinical stem-cell therapy is likely to be performed with immature progenitor cells, blood group ABO compatibility of donor cells/recipients should be favorable to avoid unnecessary rejection problems caused by ABO incompatibility. The in vitro loss of B antigens in a genotype B hESC line indicates that loss of ABH antigens occurs early during human embryogenesis since these antigens are lacking in adult cardiomyocytes.
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| 10. |
- Norström, Anders, et al.
(författare)
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Molecular and pharmacological properties of human embryonic stem cell-derived cardiomyocytes.
- 2006
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Ingår i: Experimental biology and medicine (Maywood, N.J.). - 1535-3702. ; 231:11, s. 1753-62
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESCs) can be coaxed to differentiate into specific cell types, including cardiomyocyte-like cells. These cells express cardiac-specific markers and display functional similarities to their adult counterparts. Based on these properties, hESC-derived cardiomyocytes have the potential to be extremely useful in various in vitro applications and to provide the opportunity for cardiac cell replacement therapies. However, before this can become a reality, the molecular and functional characteristics of these cells need to be investigated in more detail. In the present study we differentiate hESCs into cardiomyocyte-like cells via embryoid bodies (EBs). The fraction of spontaneously beating clusters obtained from the EBs averaged approximately 30% of the total number of EBs used. These cell clusters were isolated, dissociated into single-cell suspensions, and frozen for long-term storage. The cryopreserved cells could be successfully thawed and subcultured. Using electron microscopy, we observed Z discs and tight junctions in the hESC-derived cardiomyocytes, and by immunohistochemical analysis we detected expression of cardiac-specific markers (cTnI and cMHC). Notably, using BrdU labeling we also could demonstrate that some of the hESC-derived cardiomyocytes retain a proliferative capacity. Furthermore, pharmacological stimulation of the cells resulted in responses indicative of functional adrenergic and muscarinic receptor coupling systems. Taken together, these results lend support to the notion that hESCs can be used as a source for the procurement of cardiomyocytes for in vitro and in vivo applications.
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