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Development of three-dimensional umbilical cord-derived mesenchymal stem cell cultures for differentiation into hepatocyte-like cells : apotential breakthrough in toxicological drug screening
Publication . Cipriano, Madalena; Miranda, Joana Paiva Gomes; Santos, Jorge Miguel da Silva,; Zeilinger, Katrin
A standard truly predictive in vitro hepatic model has been sought for reducing animal testing, increasing drug development efficiency and improving prediction of adverse drug reactions. The liver is the main organ responsible for xenobiotic metabolism, being particularly exposed to chemicals and its metabolites. The currently available hepatic in vitro models are insufficient. Those present low biotransformation activity, quickly lose its differentiated phenotype in vitro and/or have relevant interspecies differences. Therefore, the differentiation of stem cells (SC) into hepatocyte-like cells (HLC) has been suggested as an alternative approach to provide a representative human hepatic model. However, a functional hepatocyte-like phenotype has not yet been achieved. In this thesis, we intended to demonstrate that by (i) using a human neonatal mesenchymal stem cell (hnMSC) source, UCX®, (ii) designing a protocol to differentiate this hnMSC into HLC using the cytokines and growth factors present during liver embryogenesis and (iii) resorting to 3D culture conditions would provide an environment closer to the in vivo, and allow a more representative cellular morphology, gene expression and biological behaviour of the hepatocyte. Umbilical cord matrix hnMSC were selected for the hepatic differentiation due to its high availability, expansion capacity and genomic stability upon expansion, and, due to its more primordial origin, its higher differentiation capacity relative to other MSC sources. The hepatic differentiation protocol was step-wise developed under 2D cell culture conditions. FGF-2 at 4 ng/mL and the absence of FGF-4 resulted in an improved endoderm commitment and foregut induction during the first step of the differentiation protocol. The epigenetic modifiers trichostatin A (TSA), 5-azacytidine (5 AZA) and dimethyl sulfoxide (DMSO) were tested for an improved hepatoblast formation and maturation to HLC. The 21 day-long protocol was extended up to day 34, when a global gene expression analysis was performed. Transcriptomic analyses placed HLC between the HepG2 cell line and hpHep and distant from hnMSC and allowed us to understand how close we were from the hepatocyte transcriptome. HLC were also characterized up to day 34 at protein and functional level. DMSO at the concentration of 1 % and the cell passage day 17 of the differentiation with 24h of 5-AZA treatment, resulted in the best tested protocol. HLC were able to store glycogen, produce albumin and urea and express genes encoding for key hepatic enzymes. Most importantly, HLCs displayed stable UGTs, EROD (CY1A1/2), ECOD (CYP2B6, 1A2 and 2E1), CYP1A1, CYP2C9 and CYP3A4-dependent activities for 13 days at levels comparable to those observed in cultured primary rat hepatocytes. The presence of the hepatic drug transporters OATP-C and MRP-2 and evaluation of CYP450 activity and induction of a wide range of enzymes was observed for the first time on hnMSC derived HLCs. To further mimic the in vivo developing liver and thus achieve a more mature hepatic phenotype we established the 3D cultures. hnMSC 3D cultures aimed at evaluating hnMSC self-assembling capacity as well as the effect of this cell culture condition on its proliferation and/or differentiation potential. Viable and low proliferative hnMSC spheroids were obtained by resorting to (i) dynamic cultures using spinner flask suspension cultures (SFSC); and (ii) static cultures using ultra-low attachment (ULA) plates. In addition, 3D culture primed the hnMSC to produce a rich ECM and was successfully adapted to serum free conditions, which is particularly relevant for the hepatic differentiation. 3D culture of HLCs was established as spheroids, formed in ULA plates, and into a hollowfiber perfused bioreactor designed for high density hepatocyte culture. 3D-HLC showed a partial hepatic zonation, observed by immunofluorescence and glycogen storage staining, and an improved hepatic functionality relative to 2D-HLC, namely by converting drugs into its specific metabolites and by producing higher amounts of glutathione. A thorough evaluation of 3D-HLCs biotransformation competency was confirmed using the model drug nevirapine (NVP). 3D HLCs distinguished from 2D-HLC by producing all phase I and II metabolites and by showing the modulation of phase I, II and III enzymes expression upon 10 days of NVP treatment. HLC thilomic profile was also presented for the first time. NVP resulted in an increased glutathione synthesis and oxidation in 3D HLC indicating a higher susceptibility to NVP toxic metabolites relative to 2D-HLC. The thesis that hnMSC were capable of differentiation into HLC was here proved. By resorting to 3D culture conditions a more representative hMSC-based in vitro model of the liver physiological conditions was developed, extensively characterized and reinforced by the NVP study. This work highlights the relevance of stem cell derived HLC as an alternative model to study drug metabolism and unveil toxicity alerts associated with drug metabolism and bioactivation. The cellular system here developed is a step towards the 3 R´s implementation in terms of mechanistic studies as well as high-throughput drug screening.
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Fundação para a Ciência e a Tecnologia
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3599-PPCDT
Número da atribuição
PTDC/SAU-TOX/110457/2009