Generation of an hiPSC-Derived Co-Culture System to Assess the Effects of Neuroinflammation on Blood-Brain Barrier Integrity

The blood-brain barrier (BBB) regulates the interaction between the highly vulnerable central nervous system (CNS) and the peripheral parts of the body. Disruption of the BBB has been associated with multiple neurological disorders, in which immune pathways in microglia are suggested to play a key role. Currently, many in vitro BBB model systems lack a physiologically relevant microglia component in order to address questions related to the mechanism of BBB integrity or the transport of molecules between the periphery and the CNS. To bridge this gap, we redefined a serum-free medium in order to allow for the successful co-culturing of human inducible pluripotent stem cell (hiPSC)-derived microglia and hiPSC-derived brain microvascular endothelial-like cells (BMECs) without influencing barrier properties as assessed by electrical resistance. We demonstrate that hiPSC-derived microglia exposed to lipopolysaccharide (LPS) weaken the barrier integrity, which is associated with the secretion of several cytokines relevant in neuroinflammation. Consequently, here we provide a simplistic humanised BBB model of neuroinflammation that can be further extended (e.g., by addition of other cell types in a more complex 3D architecture) and applied for mechanistic studies and therapeutic compound profiling

Alzheimer's Risk Gene TREM2 Determines Functional Properties of New Type of Human iPSC-Derived Microglia

Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discoverySG was supported by the Roche Postdoctoral Fellowship (RPF) program and IP by the Roche Internships for Scientific Exchange (RiSE) progra

Factors affecting toxicant sensitivity of LUHMES-derived human neurons

The use of cell-based models in neurotoxicology and for neurodegenerative diseases is driven by limitations of animal data predictivity, legislative pressure and the in-creased availability of relevant cells. As all model systems cell-based in vitro models have their restrictions and limitations. For instance, the toxicological sensitivity of neuronal cells might be affected by genetic variability, differences in the metabolic state and the presence or absence of secondary cell types (e.g. astrocytes). The aim of this thesis was to characterize different factors affecting toxicity outcomes in neurons derived from a dopaminergic neuronal cell line (LUHMES: Lund human mesencephalic cells). In a first project, the conditionally immortalized cell line LUHMES was characterized with respect to changes in cellular metabolism occurring while differentiating from neuronal precursor cells to mature dopaminergic neurons. It was observed that changes in metabolic utilization and demands, i.e. the developmental state of the cells, correlate with an increase or decrease in toxicant sensitivity. The direction of the sensitivity change was defined for toxicants of different mode of action. In the second project, genomic stability and reproducibility within different subpopula-tions of this cell line were investigated. Notably, even though LUHMES have an intact set of chromosomes, a few passages in different environments (i.e. transfer to a dif-ferent lab and deposition at a cell bank) were sufficient to introduce differences be-tween subpopulations, as revealed by whole genome sequencing. These differences did not manifest as obvious genetic alterations, (i.e. larger chromosomal aberrations or mutations directly changing amino acids), but were rather founded in SNPs in non-coding regions or pleiotropic effects, which strongly influenced the toxicant sensitivity of the subpopulations. In the third project, the LUHMES cell line was used to discover stress pathways which increase cellular resilience against disturbance of the ubiquitin-proteasome system, one major hallmark of Parkinson’s disease (PD). NRF-1 was identified as a major transcription factor coordinating proteasomal recovery. Increased external glutathione (GSH) supply was found to increase NRF-1 expression and to allow cellular survival. By co-culturing LUHMES cells with astrocytes, it was observed that astro- 3 cytes are of great importance for neuronal thiol-supply and increase neuronal GSH, thereby reducing neuronal sensitivity for proteasomal inhibitors. In summary, these findings suggest that most model systems, used as basis for animal free methods, require more scientific characterization of robustness and resilience factors. By the characterization of these factors further insight into underlying mechanisms of physiological cell-cell-interactions, cellular resilience pathways as well as modes of toxicity can be discovered, and interpretation of toxicological data can be facilitated.publishe

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation

Astrocytes, the largest cell population in the human brain, are powerful inflammatory effectors. Several studies have examined the interaction of activated astrocytes with neurons, but little is known yet about human neurotoxicity under such situations and about strategies of neuronal rescue. To address this question, immortalized murine astrocytes (IMA) were combined with human LUHMES neurons and stimulated with an inflammatory (TNF, IL-1) cytokine mix (CM). Neurotoxicity was studied both in co-cultures and in monocultures after transfer of conditioned medium from activated IMA. Interventions with >20 drugs were used to profile the model system. Control IMA supported neurons and protected them from neurotoxicants. Inflammatory activation reduced this protection, and prolonged exposure of co-cultures to CM triggered neurotoxicity. Neither the added cytokines nor the release of NO from astrocytes were involved in this neurodegeneration. The neurotoxicity-mediating effect of IMA was faithfully reproduced by human astrocytes. Moreover, glia-dependent toxicity was also observed, when IMA cultures were stimulated with CM, and the culture medium was transferred to neurons. Such neurotoxicity was prevented when astrocytes were treated by p38 kinase inhibitors or dexamethasone, whereas such compounds had no effect when added to neurons. Conversely, treatment of neurons with five different drugs, including resveratrol and CEP1347, prevented toxicity of astrocyte supernatants. Thus, the sequential IMA-LUHMES neuroinflammation model is suitable for separate profiling of both glial-directed and directly neuroprotective strategies. Moreover, direct evaluation in co-cultures of the same cells allows for testing of therapeutic effectiveness in more complex settings, in which astrocytes affect pharmacological properties of neurons.publishe