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Human pluripotent stem cell-derived hepatocytes with substantially improved functionality and adult characteristics
Barbara Kuppers-Munther, Business Development Manager, Takara Bio Europe / Cellartis
Human pluripotent stem cells (hPSC) derived hepatocytes have the potential to serve as predictive human in vitro model systems for drug discovery and toxicity testing provided that they have relevant hepatic functions. However, up to now, the functionality of hPSC-derived hepatocytes has been insufficient for applications requiring high expression of multiple drug metabolizing enzymes. We have developed a novel, highly robust 2D differentiation protocol in order to obtain more functional hepatocytes from human induced pluripotent stem cells (hiPSC). The resulting hiPSC-derived hepatocytes, Enhanced hiPS-HEP, show substantial CYP1A, 2C9, 2C19, 2D6, 2E1, and 3A4 enzyme activities and have several adult hepatic characteristics, such as high expression of adult genes (e.g. CYP2C9, 2C19, 2E1, and 3A4) and low expression of fetal genes (e.g. CYP3A7 and alpha-fetoprotein). Furthermore, we will present characterization data, e.g. on phase II enzyme activities and drug transporters, as well as results from acute and chronic hepatotoxicity studies.
Importantly, we can generate homogenous hepatocyte cultures from multiple hPSC lines which show different CYP activity profiles reflecting inter-individual variation present in the population. These developments now provide a platform for an inexhaustible source of functional human hepatocytes suitable for toxicity testing and drug metabolism studies from different genetic backgrounds.
Human embryonic stem cells to model FSHD
Uli Schmidt, General Manager, Genea Biocells Pty Lyd
Facioscapulohumeral muscular dystrophy (FSHD) presents a major unmet clinical need with no specific treatments being available, and the disease mechanisms and etiology are still poorly understood at a cellular level. In order to develop an accurate human, scalable and consistent cellular disease model we created 4 human embryonic stem cell lines from donated pre-implantation genetic diagnosis (PGD) embryos carrying the FSH chromosomal deletion. We then used several rounds of high-content screening to establish a unique 3-step protocol for the differentiation of human pluripotent stem cells to skeletal muscle. To our knowledge, this is the world's first process that allows simple and robust industrial-scale cell production, achieving final yields of typically >70% without any cell sorting, selection or genetic manipulation. We investigated the muscle phenotypes from 3x FSHD, 1x Becker muscular dystrophy and 3 normal control cell lines and found consistent morphological differences in the resulting disease-affected myotubes. We further studied the molecular mechanism by RNA microarray analysis and demonstrated profound changes to genes regulating the cell cycle, myogenesis and extracellular matrix. Based on these results we are now developing a phenotypic high-content screening assay for drug development.
MESENCHYMAL STROMAL/STEM CELLS (MSCS) AND CANCER STEM CELLS (CSCS), A TANGLED INTERRELATIONSHIP WITH INTRIGUING PATHOPHYSIOLOGICAL AND CLINICAL IMPLICATIONS.
Roberto Scatena, Associate Professor, Catholic University
MSCs, a not yet well defined cell subpopulation, are extensively studied as a valuable tool for developing novel therapeutic cell-based approaches representing a typical application of the so-called regenerative medicine. The most important therapeutic proprieties of MSCs are linked to their peculiar biological activities, as:
• Promote tissue regeneration;
• Regulate immune system;
• At last but not least , a debated capability to inhibit tumor growth.
For this last property, in facts, there are conflicting and intriguing results with in-vitro studies showing anticancer activities and in vivo studies showing a clear tumor-promoting effect. These tangled data must be urgently clarified, above all considering the enormous therapeutic potential of regenerative medicine
Another cell population intensively studied, at present, are the so-called CSCs or , as defined by other Authors, tumor-maintaining cells which are a subpopulation of cancer cells that acquired some of the characteristics of stem cells to survive and adapt to ever-changing environments. Also these cells represent a not yet well defined subpopulation of cancer cells. At present, data emerge to indicate that cancer cells that resemble stem cells need not be part of the original tumor but rather may emerge during later stages of tumor development. Thereby, the observed tumor heterogeneity is probably a combination of growing genomic instability and epigenetic instability associated with the acquisition of a stem cell-like phenotype. These instability promote a new a fundamental peculiarity of CSCs, i.e., genetic plasticity. CSCs represent the ideal justification for a lot of intriguing and obscure aspects of cancer pathogenesis (i.e., cancer cell dormancy, chemoresistance, local and distant relapses).
Emerging data seems to show that MSCs could cause an inhibition of cancer cell proliferation inducing a dormant/stem like state of cancer cell. This effect could represent a dangerous drawback of regenerative medicine that must not be neglected.
Epigenetics in iPSC generation and during TGFß signaling
Stina Simonsson, Associate Professor, University of Gothenburg
Human induced pluripotent stem cells (iPSCs) are potential cell sources for; regenerative medicine, human models to unravel disease mechanism´s, and drug screening. Osteoarthritis (OA) is the most common degenerative joint disease characterized by loss of cartilage. Chondrocytes build up cartilage and several chondrocyte genes have been implicated in the onset and progression of OA. We have previously shown that chondrocytes from autologous chondrocyte implantation (ACI)-donors can be reprogrammed into iPSCs (c-iPSCs), using a footprint free method based on mRNA delivery. To be able to use c-iPSCs as models for OA, c-iPSC lines hetero-knockout of an OA associated gene were constructed by zinc finger nuclease technology, and the molecular mechanisms of directed differentiation into the chondrogenic lineage had first to be unraveled. Therefore we studied DNA methylation, expression levels of pluripotency genes and cartilage specific genes and the impact of TGF? signaling together with DNA demethylation on c-iPSCs. We show that TGF? signaling together with DNA demethylation upregulate chondrogenic early genes and eliminate pluripotency genes in c-iPSCs. We conclude that DNA demethylation may be a necessary step in the epigenetic control of differentiation of pluripotent cells into cartilage. Epigenetic events that take place during reprogramming and differentiation will be discussed.
Regulation of Endogenous Stem and Progenitor Cells by Neural-pharmacological Agents: a New Approach for Treatment of Inflammation and Fibrosis
Olga Pershina, Chief of Research Team, Siberian Division Of The Russian Academy of Medical Sciences
We discuss various possibilities use of endogenous stem cells for the degenerative diseases treatment. Each disease has a specific activity of endogenous stem cells. From our point of view, changing activity of nervous system by neural-pharmacological agents we can influence on adult stem and progenitor cells. We investigated distribution and properties of tissue-specific precursors, bone marrow stem cells of mesenchymal and hematopoietic origin. We simulated inflammation and fibrosis of the lungs, pancreas and liver by toxic agents. We investigated drugs with adrenergic, dopaminergic and serotonergic activity as potential agents for cell therapy. We have obtained new data about the role of mesenchymal and hematopoietic stem cells in the development of inflammation and fibrosis, regenerative potential of tissue-specific precursors. We determined neurotropic drugs which were able to alter the inflammation and fibrogenesis, to increase the rate of regeneration of tissue. Endogenous stem and progenitor cells were target for neurotropic drugs. Thus, we believe the modulation functions of endogenous stem cells by neural-pharmacological agents may be a promising approach in the chronic diseases treatment including diabetes and idiopathic pulmonary fibrosis.
Targeting a Monomeric Arm in the Dimerization Region of the Epidermal Growth Factor Receptor (EGFR )Family toward Innovating Novel Antitumor Compounds
Zechariah Marting, PhD , University Of Sydney
Dimerization is a common biochemical process among countless proteins leading up to specific expectations of the dimers, and in the case of the EGFR family cell proliferation, survival, etc. EGFR family is both the archetypal and single known receptor tyrosine kinase group to involve almost all its members (ErbB1, ErbB2, ErbB3 and ErbB4) in oligomerization processes, to form homo- or heterodimers. Noting the dimerization process as a key functional requirement when two of its members form a complex with any two of its cognate ligands, particularly EGFR, followed by transphosphorylation and docking of adaptor/scaffold proteins, an avalanche of biochemical processes follow that have dramatic influence on gene transcription and cytoskeletal cells. The significance of EGFR in tumorigenesis is massive hence a number of compounds have mechanistically been developed (such as the monoclonal antibodies and tyrosine kinase inhibitors), however, faced penetratingly with erupting side effects and resistance, in particular. These dispose the need for novel approaches as a necessity. Here, we target a monomeric arm of a dimer in the dimerization region through use of pioneering small molecules to block the overlapping of the two different arms of the receptor pair, especially in an aberrant state like cancer
Self-assembled Instructive Extracellular Mimics
Nilofar Faruqui, Higher Research Scientist, National Physical Laboratory
Stem cells or progenitor cells that can differentiate into any tissue appear to provide an efficient solution to the biology of tissue repair. However, questions remain regarding physicochemical aspects of differentiation, in particular, the precise role of self-assembled extracellular matrices (ECM) and their cell-instructing interactions. Over the past years, a clear shift in the use of ‘passive materials’ to ‘instructive materials’ has been seen. Biomolecular self-assembly is an efficient strategy for biomaterial construction which can be programmed to support desired function.
Here, we introduce a single-peptide self-assembly topology, which adopts a helical type of folding, which enables the assembly of fibrous matrices, microscopic and biologically differential. The described matrix is a synthetic approximation of the native extracellular matrices, which shares key physico-chemical characteristics of the native systems including nanoscale order, hyper branched and knotted morphology and high persistence length of fibrillar structures. All these properties contribute to the formation of intricate fibrous networks that span nano-to-sub-millimeter dimensions thereby allowing for the continuous expression of unique bio-functional characteristics programmed in the sequence whose biological performance, scaffold support for mammalian cell proliferation and resistance against bacterial colonization, correlates with their morphological and chemical properties promoting thus an architectural model for differential extracellular matrices (1).
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