A human stem cell-derived test system for agents modifying neuronal N-methyl-d-aspartate-type glutamate receptor Ca2+-signalling

in Archives of Toxicology (2021)

A structure-activity relationship linking non-planar PCBs to functional deficits of neural crest cells: new roles for connexins

in Archives of Toxicology (2018), 92(3), 12251247

Migration of neural crest cells (NCC) is a fundamental developmental process, and test methods to identify interfering toxicants have been developed. By examining cell function endpoints, as in the ... [more ▼]

Migration of neural crest cells (NCC) is a fundamental developmental process, and test methods to identify interfering toxicants have been developed. By examining cell function endpoints, as in the ‘migration-inhibition of NCC (cMINC)’ assay, a large number of toxicity mechanisms and protein targets can be covered. However, the key events that lead to the adverse effects of a given chemical or group of related compounds are hard to elucidate. To address this issue, we explored here, whether the establishment of two overlapping structure–activity relationships (SAR)—linking chemical structure on the one hand to a phenotypic test outcome, and on the other hand to a mechanistic endpoint—was useful as strategy to identify relevant toxicity mechanisms. For this purpose, we chose polychlorinated biphenyls (PCB) as a large group of related, but still toxicologically and physicochemically diverse structures. We obtained concentration-dependent data for 26 PCBs in the cMINC assay. Moreover, the test chemicals were evaluated by a new high-content imaging method for their effect on cellular re-distribution of connexin43 and for their capacity to inhibit gap junctions. Non-planar PCBs inhibited NCC migration. The potency (1–10 μM) correlated with the number of ortho-chlorine substituents; non-ortho-chloro (planar) PCBs were non-toxic. The toxicity to NCC partially correlated with gap junction inhibition, while it fully correlated (p < 0.0004) with connexin43 cellular re-distribution. Thus, our double-SAR strategy revealed a mechanistic step tightly linked to NCC toxicity of PCBs. Connexin43 patterns in NCC may be explored as a new endpoint relevant to developmental toxicity screening. [less ▲]

Conversion of non-proliferating astrocytes into neurogenic neural stem cells: control by FGF2 and IFN-gamma

in Stem Cells (2016), 34(12), 28612874

Conversion of astrocytes to neurons, via de-differentiation to neural stem cells (NSC), may be a new approach to treat neurodegenerative diseases and brain injuries. The signaling factors affecting such a ... [more ▼]

Conversion of astrocytes to neurons, via de-differentiation to neural stem cells (NSC), may be a new approach to treat neurodegenerative diseases and brain injuries. The signaling factors affecting such a cell conversion are poorly understood, and they are hard to identify in complex disease models or conventional cell cultures. To address this question, we developed a serum-free, strictly controlled culture system of pure and homogeneous ‘astrocytes generated form murine embryonic stem cells (ESC)’. These stem cell derived astrocytes (mAGES), as well as standard primary astrocytes resumed proliferation upon addition of FGF. The signaling of FGF receptor tyrosine kinase converted GFAP-positive mAGES to nestin-positive NSC. ERK phosphorylation was necessary, but not sufficient, for cell cycle re-entry, as EGF triggered no de-differentiation. The NSC obtained by de-differentiation of mAGES were similar to those obtained directly by differentiation of ESC, as evidenced by standard phenotyping, and also by transcriptome mapping, metabolic profiling, and by differentiation to neurons or astrocytes. The de-differentiation was negatively affected by inflammatory mediators, and in particular, interferon gamma (IFNγ) strongly impaired the formation of NSC from mAGES by a pathway involving phosphorylation of STAT1, but not the generation of nitric oxide. Thus, two antagonistic signaling pathways were identified here that affect fate conversion of astrocytes independent of genetic manipulation. The complex interplay of the respective signaling molecules that promote/inhibit astrocyte de-differentiation may explain why astrocytes do not readily form neural stem cells in most diseases. Increased knowledge of such factors may provide therapeutic opportunities to favor such conversions. [less ▲]

Functional and phenotypic differences of pure populations of stem cell-derived astrocytes and neuronal precursor cells

in Glia (2016), 64(5), 695-715

Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation ... [more ▼]

Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation. Current protocols to prepare astrocyte cultures differ in their final content of immature precursor cells, pre-activated cells or entirely different cell types. A new method taking care of all these issues would improve research on astrocyte functions. We found here that the exposure of a defined population of pluripotent stem cell-derived neural stem cells (NSC) to BMP4 results in pure, non-proliferating astrocyte cultures within 24-48 h. These murine astrocytes generated from embryonic stem cells (mAGES) expressed the positive markers GFAP, aquaporin 4 and GLT-1, supported neuronal function, and acquired innate immune functions such as the response to TNF and IL-1. The protocol was applicable to several normal or disease-prone pluripotent cell lines, and the corresponding mAGES all exited the cell cycle and lost most of their nestin expression, in contrast to astrocytes generated by serum-addition or obtained as primary cultures. Comparative gene expression analysis of mAGES and NSC allowed quantification of differences between the two cell types and a definition of an improved maker set to define astrocytes. Inclusion of several published data sets in this transcriptome comparison revealed the similarity of mAGES with cortical astrocytes in vivo. Metabolic analysis of homogeneous NSC and astrocyte populations revealed distinct neurochemical features: both cell types synthesized glutamine and citrate, but only mature astrocytes released these metabolites. Thus, the homogeneous cultures allowed an improved definition of NSC and astrocyte features. [less ▲]

State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

in ALTEX: Alternativen zu Tierexperimenten (2014), 31(4), 441-77

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk ... [more ▼]

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing. [less ▲]