Induced pluripotent stem cell (iPSC) modeling validates reduced GBE1 enzyme activity due to a novel variant, p.Ile694Asn, found in a patient with suspected glycogen storage disease IV.

Background: Glycogen Storage disease type 4 (GSD4), a rare disease caused by glycogen branching enzyme 1 (GBE1) deficiency, affects multiple organ systems including the muscles, liver, heart, and central nervous system. Here we report a GSD4 patient, who presented with severe hepatosplenomegaly and cardiac ventricular hypertrophy. GBE1 sequencing identified two variants: a known pathogenic missense variant, c.1544G>A (p.Arg515His), and a missense variant of unknown significance (VUS), c.2081T>A (p. Ile694Asn). As a liver transplant alone can exacerbate heart dysfunction in GSD4 patients, a precise diagnosis is essential for liver transplant indication. To characterize the disease-causing variant, we modeled patient-specific GBE1 deficiency using CRISPR/Cas9 genome-edited induced pluripotent stem cells (iPSCs). Methods: iPSCs from a healthy donor (iPSC-WT) were genome-edited by CRISPR/Cas9 to induce homozygous p.Ile694Asn in GBE1 (iPSC-GBE1-I694N) and differentiated into hepatocytes (iHep) or cardiomyocytes (iCM). GBE1 enzyme activity was measured, and PAS-D staining was performed to analyze polyglucosan deposition in these cells. Results: iPSCGBE1-I694N differentiated into iHep and iCM exhibited reduced GBE1 protein level and enzyme activity in both cell types compared to iPSCwt. Both iHepGBE1-I694N and iCMGBE1-I694N showed polyglucosan deposits correlating to the histologic patterns of the patient's biopsies. Conclusions: iPSC-based disease modeling supported a loss of function effect of p.Ile694Asn in GBE1. The modeling of GBE1 enzyme deficiency in iHep and iCM cell lines had multi-organ findings, demonstrating iPSC-based modeling usefulness in elucidating the effects of novel VUS in ultra-rare diseases.

Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.

Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.

Modeling Human Bile Acid Transport and Synthesis in Stem Cell-Derived Hepatocytes with a Patient-Specific Mutation.

The bile salt export pump (BSEP) is responsible for the export of bile acid from hepatocytes. Impaired transcellular transport of bile acids in hepatocytes with mutations in BSEP causes cholestasis. Compensatory mechanisms to regulate the intracellular bile acid concentration in human hepatocytes with BSEP deficiency remain unclear. To define pathways that prevent cytotoxic accumulation of bile acid in hepatocytes, we developed a human induced pluripotent stem cell-based model of isogenic BSEP-deficient hepatocytes in a Transwell culture system. Induced hepatocytes (i-Heps) exhibited defects in the apical export of bile acids but maintained a low intracellular bile acid concentration by inducing basolateral export. Modeling the autoregulation of bile acids on hepatocytes, we found that BSEP-deficient i-Heps suppressed de novo bile acid synthesis using the FXR pathway via basolateral uptake and export without apical export. These observations inform the development of therapeutic targets to reduce the overall bile acid pool in patients with BSEP deficiency.