Unconjugated bilirubin induces neuro-inflammation in an induced pluripotent stem cell-derived cortical organoid model of Crigler Najjar Syndrome

Abstract

Bilirubin-induced neurological damage (BIND), which is also known as Kernicterus, occurs as a consequence of defects in the bilirubin conjugation machinery, thus resulting in unconjugated bilirubin (UCB) to cross the blood-brain barrier (BBB) and accumulation. Severe hyperbilirubinemia can be caused by a mutation within the UGT1A1 encoding gene. This mutation has a direct contribution towards bilirubin conjugation leading to Kernicterus as a symptom of Crigler Najjar Syndromes (CNS1, CNS2) and Gilbert syndrome, which results in permanent neurological sequelae. In this comparative study, we used human induced pluripotent stem cells (hiPSCs)-derived 3D-brain organoids to model BIND in vitro and unveil the molecular basis of the detrimental effects of UCB in the developing human brain. hiPSC-derived from healthy and CNS patients were differentiated into day-20 brain organoids, these were then stimulated with 200nM UCB. Analyses at 24- and 72-hrs post-treatment point at UCB-induced neuro-inflammation in both cell lines. Transcriptome and associated KEGG and Gene Ontology analyses unveiled activation of distinct inflammatory pathways such as cytokine-cytokine receptor interaction, MAPK signaling, calcium signaling, NF{kappa}B activation. Furthermore, both mRNA expression and secretome analysis confirmed an upregulation of pro-inflammatory cytokines such as IL6 and IL8 upon UCB stimulation. In summary, this novel study has provided insights into how a human iPSC-derived 3D-brain organoid model can serve as a prospective platform for studying the etiology of BIND-Kernicterus.