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UCX® cells: A primordial stem cell source for in vitro differentiation into hepatocyte-like cells (HLCs)
S140
Abstracts / Toxicology Letters 229S (2014) S40–S252
P-2.145 UCX® cells: A primordial stem cell source for in vitro differentiation into hepatocyte-like cells (HLCs) Madalena Cipriano 1 , Alexandra Medeiros 1 , Elysse Filipe 1 , Jorge M. Santos 2 , Rita Barcia 2 , Helder Cruz 2 , Pedro Cruz 2 , Nuno G. Oliveira 1 , Joana P. Miranda 1,∗ 1 Research Institute for Medicines – iMed.ULisboa, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal, 2 ECBio S.A., Lisboa, Portugal
Current in vitro hepatic models for toxicological screening present hindrances regarding low lifespan, biotransformation activity and interspecies variation issues. Stem cell differentiation into hepatocyte-like cells (HLCs) has been extensively investigated, being the cell source still an important point balancing ethics, species origin and differentiation potential. UCX® , a homogeneous population of human mesenchymal stromal cells, isolated from the umbilical cord tissue using a patented method, were hereby sequentially exposed to growth factors and cytokines, mimicking liver embryogenesis/development, aiming to evaluate their ability to undergo hepatocyte-like differentiation. The cells differentiated, using a 21-day protocol, were evaluated regarding their morphology, gene expression of hepatic markers and metabolic activity. Undifferentiated UCX® (negative control), and primary hepatocytes (primHep) and HepG2 cell line (positive controls) were used as controls. UCX® were able to differentiate into HLCs presenting an epithelial polygonal hepatocyte-like morphology and glycogen storage ability. Moreover, expression of albumin, CK18, HNF4␣, and underexpression of CK19 was shown by RT-PCR and immunofluorescence. Urea production, in differentiated cells, was increase when compared with HepG2 and similar to primHep. Phase I and II enzymes activity was evaluated by means of ECOD and UGT activities, respectively. Undifferentiated UCX® showed no relevant ECOD or UGT activities. In contrast, differentiated UCX® exhibited ECOD activity similar to primHep, with or without 3-methylcolanthrene-induction; whereas UGT activity was significantly improved comparing to positive controls. This study showed that UCX® is a promising cell source for deriving HLCs, thus providing a suitable tool for in vitro toxicology and drug discovery applications. Acknowledgement: TOX/110457/2009.
FCT
funded
project:
PTDC/SAU-
http://dx.doi.org/10.1016/j.toxlet.2014.06.492 P-2.146 Airborne exposure of A549 air–liquid-interphase cultures to formaldehyde at low ppm by using a novel exposure incubator system Johanna Gostner 1,∗ , Johannes Zeisler 1 , Simon Überall 1 , Kathrin Becker 2 , Peter Gruber 1 , Martina Naschberger 1 , Stefan Martini 3 , Markus Kleinhappl 3 , Florian Überall 1 1 Medical Biochemistry, Medical University Innsbruck, Innsbruck, Austria, 2 Biogical Chemistry, Medical University Innsbruck, Innsbruck, Austria, 3 Bioenergy 2020+, Graz, Austria
Background: A novel exposure incubator system has been developed to investigate the effects of volatile organic compounds (VOC)
on human cells. With this equipment, long-term exposures of air–liquid-interphase (ALI) cultures to different concentrations of gaseous compounds can be realized. Aim: Performance of the system was validated by exposing ALI cultures of the human alveolar epithelial adenocarcinoma cell line A549 cells to formaldehyde (FA). Methods: Biological responses were analyzed with a transcriptomics approach. FA concentration in the gas stream was measured by Aero-Laser technology (Aero-Laser GmbH, Germany) and FA accumulated in the culture medium was determined after 2,4dinitrophenylhydrazine (DNPH) derivatisation via HPLC. Accurate maintenance of chamber and gas stream temperatures guarantees a relative humidity ≥95%, which is necessary to reduce media loss during long term experiments. Results: ALI cultures of A549 cells were exposed to 0.1–0.5 ppm FA for 72 h. Wells of a 24-well plate were filled with media, and transwell inserts containing A549 ALI cultures were placed at the middle rows. The determined liquid input into the gas stream during the exposure period was far below the theoretical value calculated for ≥95%, indicating an optimal maintenance of temperature and humidity as well as an accurate design of the incubator boxes. The amount of FA intake was significantly higher in the wells filled with medium only than in transwell containing ones, were FA intake was at the limit of detection. Thus, A549 culture surfaces were exposed to substantial amounts of airborne FA, indicated also by a typical expression profile. http://dx.doi.org/10.1016/j.toxlet.2014.06.493 P-2.147 A primary fish gill cell model to assess xenobiotic transport and bioconcentration in fish Lucy Stott 1,2,∗ , Sabine Schnell 1 , Christer Hogstrand 1 , Stewart Owen 2 , Nic Bury 1 1
King’s College London, London, UK, 2 AstraZeneca, Brixham, UK
Many thousands of fish are used in regulatory bioconcentration tests to assess the uptake of xenobiotics from the aquatic environment. In order to replace or reduce these numbers an effective and realistic in vitro screen is required. The gills of fish are continuously bathed in water and are a principle site of both xenobiotic uptake and efflux. The current study uses a two-compartment in vitro rainbow trout primary gill cell system, which forms a polarised epithelium with high transepithelial electrical resistance (TEER; >20 k cm−2 ) and low paracellular permeability, as in vivo. Importantly, the model is able to withstand apical freshwater application, and so is a useful tool in studying the behaviour of xenobiotics in freshwater and how they may cross the gill to bioconcentrate in organisms. Xenobiotic transport assays of seven pharmaceuticals showed that all were transported across the gill epithelium in both directions. Further studies at low, environmentally relevant concentrations, and using known inhibitors of drug transport proteins, revealed that for some xenobiotics, facilitated transport appears to be a route of their uptake and efflux. Crucially, in vitro transport using this primary gill cell system correlates with predicted in silico and measured in vivo plasma concentrations, further validating the use of this model as a potential complement or surrogate to refine, reduce or replace the numbers of fish used in bioconcentration studies. http://dx.doi.org/10.1016/j.toxlet.2014.06.494
Abstracts / Toxicology Letters 229S (2014) S40–S252
P-2.145 UCX® cells: A primordial stem cell source for in vitro differentiation into hepatocyte-like cells (HLCs) Madalena Cipriano 1 , Alexandra Medeiros 1 , Elysse Filipe 1 , Jorge M. Santos 2 , Rita Barcia 2 , Helder Cruz 2 , Pedro Cruz 2 , Nuno G. Oliveira 1 , Joana P. Miranda 1,∗ 1 Research Institute for Medicines – iMed.ULisboa, Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal, 2 ECBio S.A., Lisboa, Portugal
Current in vitro hepatic models for toxicological screening present hindrances regarding low lifespan, biotransformation activity and interspecies variation issues. Stem cell differentiation into hepatocyte-like cells (HLCs) has been extensively investigated, being the cell source still an important point balancing ethics, species origin and differentiation potential. UCX® , a homogeneous population of human mesenchymal stromal cells, isolated from the umbilical cord tissue using a patented method, were hereby sequentially exposed to growth factors and cytokines, mimicking liver embryogenesis/development, aiming to evaluate their ability to undergo hepatocyte-like differentiation. The cells differentiated, using a 21-day protocol, were evaluated regarding their morphology, gene expression of hepatic markers and metabolic activity. Undifferentiated UCX® (negative control), and primary hepatocytes (primHep) and HepG2 cell line (positive controls) were used as controls. UCX® were able to differentiate into HLCs presenting an epithelial polygonal hepatocyte-like morphology and glycogen storage ability. Moreover, expression of albumin, CK18, HNF4␣, and underexpression of CK19 was shown by RT-PCR and immunofluorescence. Urea production, in differentiated cells, was increase when compared with HepG2 and similar to primHep. Phase I and II enzymes activity was evaluated by means of ECOD and UGT activities, respectively. Undifferentiated UCX® showed no relevant ECOD or UGT activities. In contrast, differentiated UCX® exhibited ECOD activity similar to primHep, with or without 3-methylcolanthrene-induction; whereas UGT activity was significantly improved comparing to positive controls. This study showed that UCX® is a promising cell source for deriving HLCs, thus providing a suitable tool for in vitro toxicology and drug discovery applications. Acknowledgement: TOX/110457/2009.
FCT
funded
project:
PTDC/SAU-
http://dx.doi.org/10.1016/j.toxlet.2014.06.492 P-2.146 Airborne exposure of A549 air–liquid-interphase cultures to formaldehyde at low ppm by using a novel exposure incubator system Johanna Gostner 1,∗ , Johannes Zeisler 1 , Simon Überall 1 , Kathrin Becker 2 , Peter Gruber 1 , Martina Naschberger 1 , Stefan Martini 3 , Markus Kleinhappl 3 , Florian Überall 1 1 Medical Biochemistry, Medical University Innsbruck, Innsbruck, Austria, 2 Biogical Chemistry, Medical University Innsbruck, Innsbruck, Austria, 3 Bioenergy 2020+, Graz, Austria
Background: A novel exposure incubator system has been developed to investigate the effects of volatile organic compounds (VOC)
on human cells. With this equipment, long-term exposures of air–liquid-interphase (ALI) cultures to different concentrations of gaseous compounds can be realized. Aim: Performance of the system was validated by exposing ALI cultures of the human alveolar epithelial adenocarcinoma cell line A549 cells to formaldehyde (FA). Methods: Biological responses were analyzed with a transcriptomics approach. FA concentration in the gas stream was measured by Aero-Laser technology (Aero-Laser GmbH, Germany) and FA accumulated in the culture medium was determined after 2,4dinitrophenylhydrazine (DNPH) derivatisation via HPLC. Accurate maintenance of chamber and gas stream temperatures guarantees a relative humidity ≥95%, which is necessary to reduce media loss during long term experiments. Results: ALI cultures of A549 cells were exposed to 0.1–0.5 ppm FA for 72 h. Wells of a 24-well plate were filled with media, and transwell inserts containing A549 ALI cultures were placed at the middle rows. The determined liquid input into the gas stream during the exposure period was far below the theoretical value calculated for ≥95%, indicating an optimal maintenance of temperature and humidity as well as an accurate design of the incubator boxes. The amount of FA intake was significantly higher in the wells filled with medium only than in transwell containing ones, were FA intake was at the limit of detection. Thus, A549 culture surfaces were exposed to substantial amounts of airborne FA, indicated also by a typical expression profile. http://dx.doi.org/10.1016/j.toxlet.2014.06.493 P-2.147 A primary fish gill cell model to assess xenobiotic transport and bioconcentration in fish Lucy Stott 1,2,∗ , Sabine Schnell 1 , Christer Hogstrand 1 , Stewart Owen 2 , Nic Bury 1 1
King’s College London, London, UK, 2 AstraZeneca, Brixham, UK
Many thousands of fish are used in regulatory bioconcentration tests to assess the uptake of xenobiotics from the aquatic environment. In order to replace or reduce these numbers an effective and realistic in vitro screen is required. The gills of fish are continuously bathed in water and are a principle site of both xenobiotic uptake and efflux. The current study uses a two-compartment in vitro rainbow trout primary gill cell system, which forms a polarised epithelium with high transepithelial electrical resistance (TEER; >20 k cm−2 ) and low paracellular permeability, as in vivo. Importantly, the model is able to withstand apical freshwater application, and so is a useful tool in studying the behaviour of xenobiotics in freshwater and how they may cross the gill to bioconcentrate in organisms. Xenobiotic transport assays of seven pharmaceuticals showed that all were transported across the gill epithelium in both directions. Further studies at low, environmentally relevant concentrations, and using known inhibitors of drug transport proteins, revealed that for some xenobiotics, facilitated transport appears to be a route of their uptake and efflux. Crucially, in vitro transport using this primary gill cell system correlates with predicted in silico and measured in vivo plasma concentrations, further validating the use of this model as a potential complement or surrogate to refine, reduce or replace the numbers of fish used in bioconcentration studies. http://dx.doi.org/10.1016/j.toxlet.2014.06.494