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Heidelberg, Germany

Peitz M.,Stem Cell Engineering Group at the Institute of Reconstructive Neurobiology | Peitz M.,University of Bonn | Munst B.,Stem Cell Engineering Group at the Institute of Reconstructive Neurobiology | Munst B.,University of Bonn | And 8 more authors.
Stem Cell Research | Year: 2014

A comprehensive understanding of the functional network of transcription factors establishing and maintaining pluripotency is key for the development of biomedical applications of stem cells. Nanog plays an important role in early development and is essential to induce natural pluripotency in embryonic stem cells (ESCs). Inducible gain-of-function systems allowing a precise control over time and dosage of Nanog activity would be highly desirable to study its vital role in the establishment and maintenance of pluripotency at molecular level. Here we engineered a recombinant cell permeable version of Nanog by fusing it with the cell penetrating peptide TAT. Nanog-TAT can be readily expressed in and purified from E. coli and binds to a consensus Nanog DNA sequence. At cellular level it enhances proliferation and self-renewal of ESCs in the absence of leukemia inhibitory factor (LIF). Nanog-TAT together with LIF acts synergistically as judged by enhanced clonogenicity and activation of an Oct4-promoter-driven GFP reporter gene. Furthermore Nanog-TAT, in the absence of LIF, promotes pluripotency by inhibiting endodermal specification in a Stat3-independent manner. Our results demonstrate that Nanog protein transduction is an attractive tool allowing control over dose and time of addition to the cells for studying the molecular control of pluripotency without genetic manipulation. © 2014.

Medicyte Gmbh | Date: 2011-10-04

The invention relates to a method for carrying out genotoxicity tests of chemical, biological and physical active substances or agents with the aid of cell culture systems of proliferating physiologically active liver cells.

Levy G.,Hebrew University of Jerusalem | Bomze D.,Hebrew University of Jerusalem | Heinz S.,Upcyte Technologies GmbH | Ramachandran S.D.,Medicyte GmbH | And 7 more authors.
Nature Biotechnology | Year: 2015

Hepatocytes have a critical role in metabolism, but their study is limited by the inability to expand primary hepatocytes in vitro while maintaining proliferative capacity and metabolic function. Here we describe the oncostatin M (OSM)-dependent expansion of primary human hepatocytes by low expression of the human papilloma virus (HPV) genes E6 and E7 coupled with inhibition of epithelial-to-mesenchymal transition. We show that E6 and E7 expression upregulates the OSM receptor gp130 and that OSM stimulation induces hepatocytes to expand for up to 40 population doublings, producing 1013 to 1016 cells from a single human hepatocyte isolate. OSM removal induces differentiation into metabolically functional, polarized hepatocytes with functional bile canaliculi. Differentiated hepatocytes show transcriptional and toxicity profiles and cytochrome P450 induction similar to those of primary human hepatocytes. Replication and infectivity of hepatitis C virus (HCV) in differentiated hepatocytes are similar to those of Huh7.5.1 human hepatoma cells. These results offer a means of expanding human hepatocytes of different genetic backgrounds for research, clinical applications and pharmaceutical development. © 2015 Nature America, Inc.

Norenberg A.,Medicyte GmbH | Heinz S.,Medicyte GmbH | Scheller K.,Medicyte GmbH | Hewitt N.J.,Medicyte GmbH | And 2 more authors.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2013

"Upcyte® human hepatocytes" have the unique property of combining proliferation with the expression of drug metabolising activities. In our current study, we evaluated whether these cells would be suitable for early in vitro micronucleus (MN) tests. A treatment period of 96h without a recovery period was most reliable for detecting MN formation in upcyte® hepatocytes from Donor 740. The basal MN rate in upcyte® hepatocytes varied considerably between donors (7-28%); therefore, modifications to the assay medium were tested to determine whether they could decrease inherent MN formation. Optimal medium supplements were 10ng/ml oncostatin M for the pre-culture and recovery periods and 25ng/ml epidermal growth factor and 10ng/ml oncostatin M for the treatment period. Using the optimised conditions and outcome criteria, the upcyte® hepatocyte MN assay could correctly identify directly acting (e.g. mitomycin C, etoposide) and metabolically activated genotoxins (e.g. benzo[a]pyrene, cyclophosphamide). "True negative" and "false positive" compounds were also correctly identified as negative. The basal %MN in upcyte® hepatocytes from Donor 740 treated with DMSO, cyclophosphamide or MMC, was essentially unaffected by the growth stage ranging from population doublings of 14-61, suggesting that billions of cells could be produced from a single donor for standardised drug toxicity testing.In conclusion, we have established and optimised an in vitro MN test by using upcyte® hepatocytes to correctly identify known direct and metabolically activated genotoxicants as well as "false positives" and true negative compounds. The almost unlimited supply of cells from a single donor and optimised test conditions increase reproducibility in early and more predictive in vitro MN tests. © 2013 Elsevier B.V.

Ramachandran S.D.,Medicyte GmbH | Schirmer K.,University of Heidelberg | Munst B.,Medicyte GmbH | Heinz S.,Medicyte GmbH | And 10 more authors.
PLoS ONE | Year: 2015

In this study we used differentiated adult human upcyte cells for the in vitro generation of liver organoids. Upcyte1 cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte process). Proliferating upcyte1 cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte1 cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel, they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions. © 2015 Ramachandran et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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