Predict Drug-Induced Liver Injury With Greater Confidence

PROGRAMMED HUMAN IPSC-DERIVED HEPATOCYTES AS A ROBUST AND HIGHLY CONSISTENT NEW APPROACH METHODOLOGY (NAM) FOR TRANSLATIONAL RESEARCH AND TOXICITY TESTING. Authors *Poster presenter (gianmarco.mastrogiovanni@bit.bio) 4. ioHepatocytes express key hepatocyte functional transcripts 5. ioHepatocytes express Phase I, II and III metabolism genes similar to PHH and HepaRG cells G. Mastrogiovanni * S. Ghimire J. Hundling V. Cornelius R. Piscupescu V. Janus L. Kowalski S. Milde A. Knights V. Yianni T. Harris-Brown N. James B. Newman K. Firth W. Bernard Hepatocytes 10-4 10-3 10-2 10-1 100 101 102 103 104 0 50 100 150 Concentration (uM) % Viability (relative to vehicle control) ioHepatocytes 24h treatment Acetaminophen Amiodarone Atorvastatin Sunitinib Ketokonazole Paroxetine Valproic acid 10-4 10-3 10-2 10-1 100 101 102 103 104 0 50 100 150 Concentration (uM) % Viability (relative to vehicle control) PHH 24h treatment Phase I metabolism – CYP450 enzymes Phase II and III metabolism A C E Albumin Bile Canaliculi Formation B a Lipid Accumulation A Hepatocyte marker expressionPluripotency markerA Albumin Secretion Ammonia Accumulation 2% CDL – 10% CDL – 20% CDL Glycogen StorageD PAS Staining – 0 min – 60 min 10-4 10-3 10-2 10-1 100 101 102 103 104 0 50 100 150 Concentration (uM) % Viability (relative to vehicle control) PHH 24h treatment Acetaminophen Amiodarone Atorvastatin Sunitinib Ketokonazole Paroxetine Valproic acid 10-4 10-3 10-2 10-1 100 101 102 103 104 0 50 100 150 Concentration (uM) % Viability (relative to vehicle control) PHH 24h treatment Acetaminophen Amiodarone Atorvastatin Sunitinib Ketokonazole Paroxetine Valproic acid 10-4 10-3 10-2 10-1 100 101 102 103 104 0 50 100 150 Concentration (uM) % Viability (relative to vehicle control) PHH 24h treatment Acetaminophen Amiodarone Atorvastatin Sunitinib Ketokonazole Paroxetine Valproic acid 10-4 10-3 10-2 10-1 100 101 102 103 104 0 50 100 150 Concentration (uM) % Viability (relative to vehicle control) PHH 24h treatment Acetaminophen Amiodarone Atorvastatin Sunitinib Ketokonazole Paroxetine Valproic acid ioHepatocytes 24h treatment PHH 24h treatment B A) Plots showing activity for CYP3A using the Promega CYP-gloTM assay at different timepoints of ioHepatocyte culturing. B) Plots showing viability of ioHepatocytes and primary human hepatocytes (PHH) after 24 hours of exposure to drugs known to cause drug induced liver injury (DILI). Data shows similar cytotoxic response in both cell types. No response was observed in either ioHepatocytes and PHH after treatment with Valproic Acid (known to not cause cytotoxicity in vitro) and Acetaminophen (which typically requires a higher dose to cause cytotoxicity). CYP3A4 Activity RLU/million cells/24 hrs Day 11 Day 17 1x107 8x106 6x106 4x106 2x106 Abstract The transition towards New Approach Methodologies (NAMs) in preclinical drug development is driven by an urgent need for human- relevant models that accurately predict drug-induced liver injury (DILI) and reduce reliance on animal testing. Hepatocytes comprise over 80% of the liver mass and handle critical functions like lipid and glucose metabolism, protein secretion, detoxification, and xenobiotic metabolism. While primary human hepatocytes (PHHs) are the gold standard in vitro model for NAMs, their utility is restricted by batch-to-batch variability and poor long-term survival. Conversely, immortalised cell lines possess physiological discrepancies when compared to PHHs. Here, we used opti-ox™, a deterministic cell programming technology, to generate human pluripotent stem cell (hiPSC)-derived hepatocytes, termed ioHepatocytes, with the consistency and scalability required for a robust, human-centric NAM. ioHepatocytes display classic cobblestone morphology with distinctive nuclei. Cells express key pan-hepatocyte markers, including ALB, HNF4A, and SERPINA1, and present a transcriptomic signature similar to PHHs. Furthermore, ioHepatocytes demonstrate high consistency, a crucial prerequisite for standardised NAMs. Functionally, ioHepatocytes perform albumin secretion, glycogen storage, ammonia clearance, and lipid accumulation. Crucially, ioHepatocytes express genes involved in phase I, II, and III drug metabolism and possess functional cytochrome P450 enzymes (CYP3A, CYP2B6, and CYP1A2). When challenged with known hepatotoxins, ioHepatocytes show a dose-dependent decrease in cell viability strongly correlating with the clinical DILI severity of the compounds. Importantly, the toxic response mimics that of PHHs, validating their predictive capability. ioHepatocytes offer a consistent, functional, and scalable model, overcoming existing limitations, and improving DILI risk assessment in drug development. Expression of genes involved in Phase I – Cytochrome P450 enzymes (left) and Phase II and III (right) drug metabolism. Bulk RNA seq was performed on ioHepatocytes, HepG2, HepaRG (after 8 days in culture) and primary human hepatocytes (PHH) from 4 donors after 3 days in culture . Data from fetal liver was sourced externally and used for comparison. Data show that ioHepatocytes have a high similarity in expression profile with primary human hepatocytes. Specifically, ioHepatocytes cluster closely with PHH and HepaRG and separately from the more immature HepG2 and fetal liver tissue. Importantly, ioHepatocytes express the key enzymes CYP3A4, CYP2B6, CYP2C9, GSTA1/2 and UGT1A1, ABCB11 and ABCC2. This data show that ioHepatocytes Tox have the potential of being used as a model to assess toxicity of drugs in vitro. Downregulation (A) of pluripotency (NANOG), and upregulation (B) of G6PC (role in gluconeogenesis), ARG1 (role in urea cycling) and ALB following 4 days of programming to a hepatocyte fate. AFP (alpha-fetoprotein) is typically a fetal hepatocyte marker and demonstrates reduced expression following programming to a hepatocyte fate. Donor 1 iPSCs HepG2 Fetal Liver tissue HepaRG Donor 2 Donor 3 Donor 4 Primary Human Hepatocytes 2. ioHepatocytes display cobblestone morphology, binucleation, and expression of pan-hepatocyte markers DAPI Albumin HNF4a Albumin + HNF4a * * * * * A) Brightfield image of ioHepatocytes after 9 days in culture post-thaw. Following revival, ioHepatocytes rapidly acquire a highly homogeneous and characteristic cobblestone morphology, displaying well-defined cell borders, and frequent binucleation indicated with (*). These features represent hallmark morphological traits of mature human hepatocytes and demonstrate the robust recovery and stability of the culture following thawing. B) Immunofluorescence staining demonstrates that the cells homogeneously co-express key pan-hepatocyte markers, including Albumin (orange) and HNF4α (green), both of which are consistently maintained throughout the culture period. DAPI was used as a nuclear counterstain (blue) to visualize cell nuclei. Representative examples of binucleated cells are highlighted with (*). A B • opti-ox enabled transcription factor expression leads to the consistent generation of defined, functional human iPSC- derived hepatocytes that are ready for experiments within days and can be used reliably over 17 days in culture. • ioHepatocytes are a highly homogeneous population of cells expressing key hepatocytes markers, like ALB, HNF4A, ASGR1 and CYP3A4. Over time in culture, they downregulate fetal markers and increase expression of maturation and functionality markers. • These cells demonstrate mature hepatocyte functions, including albumin secretion, robust CYP3A4 activity, dose- dependent lipid accumulation, glycogen storage, ammonia clearance and bile canaliculi formation. • Furthermore, ioHepatocytes exhibit drug metabolism profiles comparable to PHH and HepaRG, alongside similar cytotoxic responses to DILI-inducing compounds. • Thanks to their ability to be metabolically functional (lipid, glucose and drug metabolism among others) ioHepatocytes are a valuable and reliable in vitro suitable for drug discovery, translational research, and predictive toxicology testing.