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, NFkB activation. Furthermore, both mRNA expression and secretome analysis confirmed an upregulation of proinflammatory 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.