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Updated: Aug 8, 2026
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
Bilirubin-induced immunostimulant effects and toxicity vary with neural cell type and maturation state
Ana S Falcão1, Adelaide Fernandes, Maria A Brito
1Centro de Patogénese Molecular-UBMBE, Faculdade de Farmácia, University of Lisbon, Av. Forças Armadas, 1600-083, Lisbon, Portugal.
Insights
Immature nerve cells are more susceptible to brain damage from unconjugated bilirubin (UCB). Astrocytes show higher inflammatory responses and glutamate release when exposed to UCB, contributing to understanding neonatal brain injury.
Area of Science:
- Neuroscience
- Neonatal Medicine
- Toxicology
Background:
- Neonatal hyperbilirubinemia is common, with premature infants at higher risk for brain damage.
- Unconjugated bilirubin (UCB) toxicity in immature neural cells is a significant concern.
Purpose of the Study:
- To investigate how neural cell differentiation affects vulnerability to UCB.
- To compare UCB responses in neurons and astrocytes to identify susceptible cell types.
- To explore the role of NF-kappaB activation in UCB-induced inflammation.
Main Methods:
- Cultured astrocytes and neurons at different differentiation stages were exposed to UCB.
- UCB-induced cell death, glutamate release, and cytokine production were measured.
- Nuclear factor-kappaB (NF-kappaB) activation was assessed in relation to cell age and type.
Main Results:
- Immature nerve cells exhibited greater vulnerability to UCB-induced cell death, glutamate release, and TNF-alpha secretion.
- Astrocytes demonstrated higher glutamate release and inflammatory responses to UCB injury.
- NF-kappaB activation by UCB showed cell-age and cell-type dependency, with astrocytes having the highest levels.
Conclusions:
- Neural cell differentiation significantly influences vulnerability to UCB toxicity.
- Astrocytes play a key role in UCB-induced neuroinflammation and glutamate excitotoxicity.
- Understanding age- and type-specific responses is crucial for elucidating UCB encephalopathy mechanisms.
Abstract:
Hyperbilirubinemia remains one of the most frequent clinical diagnoses in the neonatal period. The increased vulnerability of premature infants to unconjugated bilirubin (UCB)-induced brain damage may be due to a proneness of immature nerve cells to UCB-toxic stimulus. Thus, in this study, we evaluated UCB-induced cell death, glutamate release and cytokine production, in astrocytes and neurons cultured for different days, in order to relate the differentiation state with cell vulnerability to UCB. The age-dependent activation of the nuclear factor-kappaB (NF-kappaB), an important transcription factor involved in inflammation, was also investigated. Furthermore, responsiveness of neurons and astrocytes to UCB were compared in order to identify the most susceptible to each induced effect, as an approach to what happens in vivo. The results clearly showed that immature nerve cells are more vulnerable than the most differentiated ones to UCB-induced cell death, glutamate release and tumour necrosis factor (TNF)-alpha secretion. Moreover, astrocytes seem to be more competent cells in releasing glutamate and in producing an inflammatory response when injured by UCB. Activation of NF-kappaB by UCB also presents a cell-age-dependent pattern, and values vary with neural cell type. Again, astrocytes have the highest activation levels, which are correlated with the greater amount of cytokine production observed in these cells. These results contribute to a better knowledge of the mechanisms leading to UCB encephalopathy by elucidation of age- and type-related differences in neural cell responses to UCB.
