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Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing
Published on: June 9, 2017
Cytokine Profiling in Human iPSC-Derived Dopaminergic Neuronal and Microglial Cultures
Evelyn Knappe1, Franziska Rudolph1, Christine Klein1
1Institute of Neurogenetics, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany.
Abstract:
Aside from the degeneration of dopaminergic neurons, inflammation is a key component in the movement disorder Parkinson's disease (PD). Microglia activation as well as elevated cytokine levels were observed in the brains of PD patients, but the specific role of microglia in the disease process is unknown. Here, we generate human cellular models by differentiating iPSCs into dopaminergic neurons and microglia. We combine these cells in co-culture to perform cytokine profiling, representing the final functional outcome of various signaling pathways. For this, we used unstimulated conditions and treatment with inflammatory stressors. Importantly, only co-cultures but not the monocultures responded to IL-1β treatment suggesting co-culture-related crosstalk. Moreover, we identified the main types of released cytokines and chemokines in this model system and found a preference for the activation of the chemotaxis pathway in response to all treatments, which informs future studies on the cell-type-specific reaction to inflammatory stimulation. Finally, we detected protein level changes in PD risk factor GPNMB upon stress in microglia, further confirming the link between PD-associated genes and inflammation in human-derived cellular models.
Insights
Inflammation and microglia activation are key in Parkinson's disease (PD). This study developed human cell models to show microglia and neuron interactions, revealing inflammatory pathways and PD gene links.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Inflammation, particularly microglia activation, is a significant factor in Parkinson's disease (PD) pathogenesis, alongside dopaminergic neuron degeneration.
- The precise role of microglia in PD progression remains unclear, necessitating advanced cellular models for investigation.
Purpose of the Study:
- To create and utilize human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons and microglia co-culture models.
- To investigate the inflammatory responses and cytokine profiles in these co-cultures under various stimulation conditions.
- To explore the interplay between microglia and neurons in the context of PD-related inflammation.
Main Methods:
- Generation of human dopaminergic neurons and microglia from iPSCs.
- Co-culturing these differentiated cells to mimic brain microenvironments.
- Cytokine profiling using unstimulated and inflammatory-stimulated conditions, including IL-1β treatment.
- Analysis of protein level changes in PD risk factors like GPNMB.
Main Results:
- Co-cultures exhibited a significant crosstalk response to IL-1β treatment, unlike monocultures, indicating cell-cell communication.
- The chemotaxis pathway was predominantly activated across all treatment conditions, highlighting a key inflammatory response.
- Microglia showed altered protein levels of the PD risk factor GPNMB upon inflammatory stress.
Conclusions:
- Human iPSC-derived co-culture models effectively recapitulate PD-related neuroinflammation.
- Microglia-neuron interactions are crucial for understanding inflammatory responses in PD.
- These findings link PD-associated genes to inflammatory processes in human cellular models, paving the way for targeted therapies.
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