Integration of Kupffer cells into human iPSC-derived liver organoids for modeling liver dysfunction in sepsis.

Maximizing the potential of human liver organoids (LOs) for modeling human septic liver requires the integration of innate immune cells, particularly resident macrophage Kupffer cells. In this study, we present a strategy to generate LOs containing Kupffer cells (KuLOs) by recapitulating fetal liver hematopoiesis using human induced pluripotent stem cell (hiPSC)-derived erythro-myeloid progenitors (EMPs), the origin of tissue-resident macrophages, and hiPSC-derived LOs. Remarkably, LOs actively promote EMP hematopoiesis toward myeloid and erythroid lineages. Moreover, supplementing with macrophage colony-stimulating factor (M-CSF) proves crucial in sustaining the hematopoietic population during the establishment of KuLOs. Exposing KuLOs to sepsis-like endotoxins leads to significant organoid dysfunction that closely resembles the pathological characteristics of the human septic liver. Furthermore, we observe a notable functional recovery in KuLOs upon endotoxin elimination, which is accelerated by using Toll-like receptor-4-directed endotoxin antagonist. Our study represents a comprehensive framework for integrating hematopoietic cells into organoids, facilitating in-depth investigations into inflammation-mediated liver pathologies.

Stabilized generation of human iPSC-derived liver organoids using a modified coating approach.

Human-induced pluripotent stem cell (hiPSC)-derived hepatic cells are useful tools for regenerative medicine, and various culture substrates are currently used for their differentiation. We differentiated hiPSC-derived hepatic endoderm (HE), endothelial cells (ECs), and mesenchymal cells (MCs) using Laminin-511 (LN) coating to generate liver organoids, hiPSC-liver buds (hiPSC-LBs), which exhibited therapeutic effects when transplanted into disease model animals. Stably producing significant amounts of hiPSC-LBs is necessary for sufficient therapeutic effects. However, general precoating (standard coating) requires quick manipulation, often causing failure for inexperienced cell cultures, we thus tested direct LN addition to the culture medium (Direct coating). Using quantitative gene expression, flow cytometry, albumin secretion, and ammonia metabolism, we demonstrated that Standard and Direct coating similarly induce hiPSC-derived hepatocyte, mesodermal cell, EC, and MC differentiation. Standard and Direct coating-differentiated cells generated iPSC-LBs with equivalent hepatic functions. Furthermore, Direct coating enabled stable induction of differentiation independent of individual culture skills and reduced total amount of LN use as the same differentiated cell quality can be obtained upon LN supplementation at lower concentrations. In summary, the results of this study suggest that Direct coating could enable stable hiPSC-LB production at a low cost, thereby yielding mass cell production using hiPSCs.

A combination of nuclear ß-catenin and atypical scores as useful diagnostic markers for borderline malignancy of gastric tumours.

AIMS: Although recent advances in endoscopic technology, including submucosal dissection (ESD), contribute to significant improvement in detection and treatment for early gastric carcinomas (GCas), discrepant diagnosis between forceps biopsied and ESD sections sometimes occurs. Here, we focused on histological markers for accurate diagnosis of gastric tumours in forceps biopsied samples before ESD treatment. METHODS AND RESULTS: A total of 136 cases of gastric tumours, including 25 adenomas and 111 GCas, were investigated using a combination of forceps biopsied and ESD samples. Atypical scores based on both nuclear and branching parameters could distinguish between adenomas and GCas in both samples. The labelling indices (LIs) of nuclear ß-catenin were significantly higher in GCas than adenomas, and positively correlated with atypical scores, as well as branching parameters. In addition, nuclear ß-catenin immunoreactivity showing small cluster pattern was colocalized with immunoreactivity for aldehyde dehydrogenase 1 (ALDH1), known as a cancer stem cell (CSC) marker, which suggests the presence of some CSC population, and may be due to formation of dynamic tubular branching in GCas. CONCLUSIONS: A combination of nuclear ß-catenin and atypical scores may be useful for differential diagnosis of borderline malignancy of gastric tumours.