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Thyroid hormones (THs) are crucial regulators of human metabolism and early development. During the safety assessment of plant protection products, the human relevance of chemically induced TH perturbations observed in test animals remains uncertain. European regulatory authorities request follow-up in vitro studies to elucidate human-relevant interferences of thyroid gland function, or TH catabolism through hepatic enzyme induction. However, human in vitro assays, based on single molecular initiating events, poorly reflect the complex TH biology and related liver-thyroid axis. To address this complexity, we present human three-dimensional thyroid and liver organoids with key functions of TH metabolism. The thyroid model resembled in vivo-like follicular architecture and a TSH-dependent triiodothyronine synthesis over 21 days which was inhibited by methimazole. The HepaRG-based liver model, secreting critical TH-binding proteins albumin and thyroxine-binding globulin (TBG), emulated an active TH catabolism via the formation of glucuronidated and sulfated thyroxine (gT4/sT4). Activation of the nuclear receptors PXR and AHR was demonstrated via the induction of specific CYP isoenzymes by rifampicin, pregnenolone-16a-carbonitrile and β-naphthoflavone. However, this nuclear receptor activation, assumed to regulate UDP-glucuronosyltransferases and sulfotransferases, appeared to have no effect on gT4 and sT4 formation in this human-derived hepatic cell line model. Finally established single-tissue models were successfully co-cultured in a perfused two-organ chip for 21 days. In conclusion, this model presents a first step towards a complex multimodular human platform, which will help to identify both direct and indirect thyroid disruptors that are relevant from a human safety perspective.

BackgroundGiven regenerative therapies, the utilization of primary human cells is desired and requested in the development of in vitro systems and disease models. After a few passages in vitro, all cells from the connective tissue end up in a similar fibroblastoid cell type marked by loss of the specific expression pattern. It is still under discussion whether different de-differentiated mesenchymal cells have similar or identical differentiation capacities in vitro.MethodsChondrocytes isolated from patients with late-stage osteoarthritis were cultured for several passages until de-differentiation was completed. The mRNA level of cartilage markers was investigated, and the adipogenic, osteogenic and chondrogenic differentiation capacity was examined. By adding 5-aza-2’-deoxycytidine (5-aza-dC) to the media, the influence of DNA methylation on the differentiation capacity was analyzed.ResultsThe chondrocytes used in this work were not affected by the loss of specific gene expression upon cell culture. The mRNA levels of SOX5, SOX6, SOX9, aggrecan, and proteoglycan-4 remained unchanged. The underlying mechanisms of cartilage marker maintenance in osteoarthritic (OA) chondrocytes were investigated with a focus on the epigenetic modification by DNA methylation. The treatment of de-differentiated chondrocytes with the DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) displayed no appreciable impact on the observed maintenance of marker gene expression, while the chondrogenic differentiation capacity was compromised. On the other hand, the pre-cultivation with 5-aza-dC improved the osteogenesis and adipogenesis of OA chondrocytes. Contradictory to these effects, the DNA methylation levels were not reduced after treatment with 1 μM 5-aza-dC for four weeks.ConclusionChondrocytes isolated from late-stage osteoarthritic patients represents a reliable cell source for in vitro studies as wells as disease models since the chondrogenic differentiation potential remains. 5-aza-2’-deoxycytidine could not further improve their chondrogenic potential.

Background Filamentous fungi can each produce dozens of secondary metabolites which are attractive as therapeutics, drugs, antimicrobials, flavour compounds and other high-value chemicals. Furthermore, they can be used as an expression system for eukaryotic proteins. Application of most fungal secondary metabolites is, however, so far hampered by the lack of suitable fermentation protocols for the producing strain and/or by low product titers. To overcome these limitations, we report here the engineering of the industrial fungus Aspergillus niger to produce high titers (up to 4,500 mg • l−1) of secondary metabolites belonging to the class of nonribosomal peptides. Results For a proof-of-concept study, we heterologously expressed the 351 kDa nonribosomal peptide synthetase ESYN from Fusarium oxysporum in A. niger. ESYN catalyzes the formation of cyclic depsipeptides of the enniatin family, which exhibit antimicrobial, antiviral and anticancer activities. The encoding gene esyn1 was put under control of a tunable bacterial-fungal hybrid promoter (Tet-on) which was switched on during early-exponential growth phase of A. niger cultures. The enniatins were isolated and purified by means of reverse phase chromatography and their identity and purity proven by tandem MS, NMR spectroscopy and X-ray crystallography. The initial yields of 1 mg • l−1 of enniatin were increased about 950 fold by optimizing feeding conditions and the morphology of A. niger in liquid shake flask cultures. Further yield optimization (about 4.5 fold) was accomplished by cultivating A. niger in 5 l fed batch fermentations. Finally, an autonomous A. niger expression host was established, which was independent from feeding with the enniatin precursor d-2-hydroxyvaleric acid d-Hiv. This was achieved by constitutively expressing a fungal d-Hiv dehydrogenase in the esyn1-expressing A. niger strain, which used the intracellular α-ketovaleric acid pool to generate d-Hiv. Conclusions This is the first report demonstrating that A. niger is a potent and promising expression host for nonribosomal peptides with titers high enough to become industrially attractive. Application of the Tet-on system in A. niger allows precise control on the timing of product formation, thereby ensuring high yields and purity of the peptides produced. Electronic supplementary material The online version of this article (doi:10.1186/s40694-014-0004-9) contains supplementary material, which is available to authorized users.