Abstract

Background: We and others have shown that rare bi-allelic loss-of-function mutations in Kinesin family member 12 (KIF12) cause pediatric high-GGT cholestasis as well as fibrosis, ductular reaction, and bile duct loss. KIF12 has putatively been defined as a microtubule-associated motor protein, but little is known about its function and even less about its role in the pathogenesis of cholestasis.

Methods: We used Crispr-Cas9 technology to introduce a homozygous Arg219 premature termination mutation (Arg219*) reported in 6 patients into a human induced pluripotent stem cell (hiPSC) line. Wild-type and Arg219* hiPSCs were differentiated into cholangiocyte-like cells (iCCs) using a protocol that induces definitive endoderm followed by hepatic endoderm, hepatoblast, and finally iCCs. These iCCs were plated as monolayers, polarized on transwells, and cultured as 3D basolateral-out and apical-out organoids. Confocal microscopy was used to visualize protein markers and organelle localization. GGT activity and secretin response were performed. qPCR and bulk RNA sequencing were used to identify transcriptomic signatures.

Results:: iPSC differentiated cholangiocytes display stage specific markers in both wild-type and Arg219* cells. Arg219* cells showed no detectable defects in growth rate, actin cytoskeleton or iCCs maturation markers but demonstrated increased GGT activity. RNA sequencing between KIF12 mutant and wild-type iCCs identified differential expression in ~100 genes with gene ontology suggesting underrepresentation of proteins with extracellular signaling domains (p = 2.5*10^-8) in KIF12 mutant iCCs. Confocal microscopy demonstrated gross organelle localization defects with decreased mitochondrial footprint (p = 1.8*10^-5), decreased lysosomal distance to nucleus (p < 2.2*10^-16), and defects in cilia positioning in KIF12 mutant iCCs as compared to wild-type iCCs controls. To better understand cilia defects we performed 3D organoid culturing. Organoids also demonstrated mature cholangiocyte markers such as CK7 and CK19 as well as functional capability as demonstrated by secretin swelling protocol. Cilia were present on organoids but exhibited mislocalization to the basolateral membrane in Arg219* iCCs, independently of membrane polarization.

Conclusion: Using iPSC differentiated cholangiocytes we have demonstrated that KIF12 deficiency leads to increased GGT, transcriptional alterations with decreased extracellular signaling, and pronounced organelle mislocalization, possibly due to an impairment in organelle trafficking. This evidence implicates kinesins as critical mediators of cell homeostasis and provides new cellular and molecular insights into human cholestatic liver disease pathogenesis.