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

Bilirubin-induced neurological damage (BIND), which might progress to kernicterus, occurs as a consequence of defects in the bilirubin conjugation machinery, thus enabling albumin-unbound free bilirubin (BF) to cross the blood–brain barrier and accumulate within. A defect in the UGT1A1 enzyme-encoding gene, which is directly responsible for bilirubin conjugation, can cause Crigler–Najjar syndrome (CNS) and Gilbert’s syndrome. We used human-induced plu-ripotent stem cell (hiPSC)-derived 3D brain organoids to model BIND in vitro and unveil the molecular basis of the detrimental effects of BF in the developing human brain. Healthy and pa-tient-derived iPSCs were differentiated into day-20 brain organoids, and then stimulated with 200 nM BF. Analyses at 24 and 72 h post-treatment point to BF-induced neuro-inflammation in both cell lines. Transcriptome, associated KEGG, and Gene Ontology analyses unveiled the acti-vation of distinct inflammatory pathways, such as cytokine–cytokine receptor interaction, MAPK signaling, and NFκB activation. Furthermore, the mRNA expression and secretome anal-ysis confirmed an upregulation of pro-inflammatory cytokines such as IL-6 and IL-8 upon BF stimulation. 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 kernic-terus. The transcriptomes of brain organoids with and without bilirubin were compared by microarray analysis (Clariom-S Human Gene Expression Array, Affymetrix, Thermo Fisher Scientific).