The development of complex multicellular human in vitro systems holds great promise for modelling disease and advancing drug discovery and tissue engineering ¹ . In the liver, despite the identification of key signalling pathways involved in hepatic regeneration ^2,3 , in vitro expansion of human hepatocytes directly from fresh patient tissue has not yet been achieved, limiting the possibility of modelling liver composite structures in vitro. Here we first developed human hepatocyte organoids (h-HepOrgs) from 28 different patients. Patient-derived hepatocyte organoids sustained long-term expansion of hepatocytes in vitro and maintained patient-specific gene expression and bile canaliculus features and function of the in vivo tissue. After transplantation, expanded h-HepOrgs rescued the phenotype of a mouse model of liver disease. By combining h-HepOrgs with portal mesenchyme and our previously published cholangiocyte organoids ^4–6 , we generated patient-specific periportal liver assembloids that retain the histological arrangement, gene expression and cell interactions of periportal liver tissue, with cholangiocytes and mesenchyme embedded in the hepatocyte parenchyma. We leveraged this platform to model aspects of biliary fibrosis. Our human periportal liver assembloid system represents a novel in vitro platform to investigate human liver pathophysiology, accelerate drug development, enable early diagnosis and advance personalized medicine.

Tissue-resident lymphocytes provide organ-adapted protection against invading pathogens. Whereas their biology has been examined in great detail in various infection models, their generation and functionality in response to vaccination have not been comprehensively analyzed in humans. We therefore studied SARS-CoV-2 mRNA vaccine–specific T cells in surgery specimens of kidney, liver, lung, bone marrow, and spleen compared with paired blood samples from largely virus-naive individuals. As opposed to lymphoid tissues, nonlymphoid organs harbored significantly elevated frequencies of spike-specific CD4⁺ T cells compared with blood showing hallmarks of tissue residency and an expanded memory pool. Organ-derived CD4⁺ T cells further exhibited increased polyfunctionality over those detected in blood. Single-cell RNA-Seq together with T cell receptor repertoire analysis indicated that the clonotype rather than organ origin is a major determinant of transcriptomic state in vaccine-specific CD4⁺ T cells. In summary, our data demonstrate that SARS-CoV-2 vaccination entails acquisition of tissue memory and residency features in organs distant from the inoculation site, thereby contributing to our understanding of how local tissue protection might be accomplished.

Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, metabolic dysfunction-associated steatotic liver disease (MASLD) is gaining importance as the disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in MASLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) an increased basal MET phosphorylation and a strong downregulation of the PI3K-AKT pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a sexually dimorphic condition, with higher prevalence in men than in women. Sex differences in hepatic lipid metabolism and the modulatory role of sex hormones have been described but are still insufficiently understood. The aim of this study was to introduce the variables sex and sex hormones into a human in vitro model of hepatic steatosis. Methods: Primary human hepatocytes (PHHs) were isolated from male and female donors, treated with free fatty acids (FFA) to induce steatosis, and further exposed to physiological concentrations of estrogen, progesterone, or testosterone. Intracellular triacylglyceride (TAG) content, lipid droplet (LD) formation, FFA uptake, and very-low-density lipoprotein (VLDL) excretion were assessed. In parallel, the expression of lipid metabolism-related genes was quantified by qPCR. Results: FFA treatment induced comparable uptake and intracellular TAG storage in both sexes. However, female PHHs secreted approximately twice as many VLDL particles as male cells. Steatosis significantly increased expression of LDLR, CPT2, and PLA1A only in male PHHs. Sex hormones exerted distinct, sex-specific effects: estrogen reduced TAG accumulation in female PHHs; whereas testosterone reduced TAG in male but increased it in female PHHs after prolonged treatment. LD characterization confirmed sex- and hormone-dependent differences in lipid storage patterns. In male PHHs, progesterone promoted lipid storage and increased apoB-100 secretion, accompanied by reduced LDLR and APOA5 expression, and testosterone increased the FFA-mediated CPT2 even further. Conclusions: Sex and sex hormones distinctly shape hepatocellular lipid handling under steatotic conditions. While female PHHs demonstrated greater lipid excretion capacity, male PHHs exhibited stronger transcriptional responses. Sex-specific responses to estrogen and testosterone resembled clinical observations on sex hormone effects. These findings highlight the need to account for sex-specific differences in MASLD pathophysiology and therapeutic strategies.

The development of complex multicellular human in vitro systems holds great promise for modelling disease and progressing drug discovery and tissue engineering ¹ . In the liver, despite the identification of key signalling pathways involved in hepatic regeneration ^2,3 , in vitro expansion of human hepatocytes directly from fresh patient tissue has not yet been achieved, limiting the possibility of modelling liver composite structures in vitro . Here, we first developed human hepatocyte organoids (h-HepOrgs) from 28 different patients. Patient-derived hepatocyte organoids sustain long-term expansion of hepatocytes in vitro and maintain patient-specific gene expression, and bile canaliculi features and function of the in vivo tissue. After transplantation, expanded human hepatocyte organoids rescue the phenotype of a mouse model of liver disease. By combining human hepatocyte organoids with portal mesenchyme and our previously published cholangiocyte organoids ^4-6 , we generated patient-specific periportal liver assembloids that retain the histological arrangement, gene expression and cell interactions of the periportal liver tissue, with cholangiocytes and mesenchyme embedded in the hepatocyte parenchyma. We leveraged this platform to model aspects of biliary fibrosis. Our human periportal liver assembloid system represents a novel in vitro platform to investigate human liver pathophysiology, accelerate drug development, enable early diagnosis and progress personalized medicine.