During chronic injury, regeneration of the adult liver becomes impaired. In this context bipotent Hepatic Progenitor Cells (HPCs) become activated and can regenerate both cholangiocytes and hepatocytes. Notch and Wnt signalling during hepatic ontogeny are described, but their roles in HPC mediated liver regeneration are unclear. Here we show in human diseased liver and murine models of the ductular reaction with biliary and hepatocyte regeneration that Notch and Wnt signalling direct HPC specification within the activated myofibroblasts and macrophages HPC niche. During biliary regeneration, Numb is downregulated in HPCs, Jagged1 promotes biliary specification within HPCs. During hepatocyte regeneration, macrophage derived canonical Wnt signalling maintains Numb within HPCs, and Notch signalling is reduced promoting hepatocyte specification. This dominant Wnt state is stimulated through engulfment of hepatocyte debris by niche macrophages and can directly influence the HPCs. Macrophage Wnt3a expression in turn facilitates hepatocyte regeneration – thus exemplifying a novel positive feedback mechanism in adult parenchymal regeneration.

BACKGROUND & AIMS Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%–10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy. METHODS We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States; epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry; integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib. RESULTS Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%; χ2 = 11.61; P < .0007), time to recurrence (13.7 vs 22.7 months; P < .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI–IV; χ2 = 8.34; P < .03); SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab. CONCLUSIONS We provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets.

ObjectiveKeratin (K)19, a biliary/hepatic progenitor cell (HPC) marker, is expressed in a subset of hepatocellular carcinomas (HCC) with poor prognosis. The underlying mechanisms driving this phenotype of K19-positive HCC remain elusive.DesignClinicopathological value of K19 was compared with EpCAM, and α-fetoprotein, in a Caucasian cohort of 242 consecutive patients (167 surgical specimens, 75 needle biopsies) with different underlying aetiologies. Using microarrays and microRNA profiling the molecular phenotype of K19-positive HCCs was identified. Clinical primary HCC samples were submitted to in vitro invasion assays and to side population analysis. HCC cell lines were transfected with synthetic siRNAs against KRT19 and submitted to invasion and cytotoxicity assays.ResultsIn the cohort of surgical specimens, K19 expression showed the strongest correlation with increased tumour size (p<0.01), decreased tumour differentiation (p<0.001), metastasis (p<0.05) and microvascular invasion (p<0.001). The prognostic value of K19 was also confirmed in a set of 75 needle biopsies. Profiling showed that K19-positive HCCs highly express invasion-related/metastasis-related markers (eg, VASP, TACSTD2, LAMB1, LAMC2, PDGFRA), biliary/HPC markers (eg, CD133, GSTP1, NOTCH2, JAG1) and members of the miRNA family 200 (eg, miR-141, miR-200c). In vitro, primary human K19-positive tumour cells showed increased invasiveness, and reside in the chemoresistant side population. Functionally, K19/KRT19 knockdown results in reduced invasion, loss of invadopodia formation and decreased resistance to doxorubicin, 5-fluorouracil and sorafenib.ConclusionsGiving the distinct invasive properties, the different molecular profile and the poor prognostic outcome, K19-positive HCCs should be considered as a seperate entity of HCCs.

A distinct pattern of HPC surface markers was found between acute and chronic liver diseases. Similar to what is known from animal experiments, strong evidence has been found signifying the role of Wnt signalling in proliferation of human HPCs whereas Notch signalling is involved in biliary differentiation. These pathways can be targeted in future therapies.

In determining relative gene expression by quantitative measurements of mRNA levels using real-time quantitative PCR, internal standards such as reference genes are essential. Large-scale studies evaluating (candidate) reference genes for veterinary research have not been conducted as thoroughly as for human research, although they are equally important. Our goal was to design and evaluate a genome-wide panel of reference genes from different functional classes. First, primers were optimized using mRNA from canine cell lines and from 30 tissues of one dog as template and SYBR green as fluorescent probe. Second, the expression variation and stability of a gene within one specific tissue were determined. Prostate, kidney, mammary gland, left ventricle, and liver tissues from five to nine dogs of different breeds, sexes, ages, body weights, and disease status were used. Averaging relative stabilities over these tissues revealed the usefulness of individual genes as reference genes. Furthermore, according to expression variation of a reference gene within a specific tissue, usually two to four reference genes are sufficient. Taken together, ribosomal protein S19 (RPS19), ribosomal protein S5 (RPS5), b-2-microglobulin (B2M), and hypoxanthine phosphoribosyltransferase (HPRT) are advocated. However, the optimal set of reference genes depends on the tissue and should be selected and evaluated for each series of experiments.

Organ‐ and tissue‐level biological functions are intimately linked to microscale cell–cell interactions and to the overarching tissue architecture. Together, biofabrication and organoid technologies offer the unique potential to engineer multi‐scale living constructs, with cellular microenvironments formed by stem cell self‐assembled structures embedded in customizable bioprinted geometries. This study introduces the volumetric bioprinting of complex organoid‐laden constructs, which capture key functions of the human liver. Volumetric bioprinting via optical tomography shapes organoid‐laden gelatin hydrogels into complex centimeter‐scale 3D structures in under 20 s. Optically tuned bioresins enable refractive index matching of specific intracellular structures, countering the disruptive impact of cell‐mediated light scattering on printing resolution. This layerless, nozzle‐free technique poses no harmful mechanical stresses on organoids, resulting in superior viability and morphology preservation post‐printing. Bioprinted organoids undergo hepatocytic differentiation showing albumin synthesis, liver‐specific enzyme activity, and remarkably acquired native‐like polarization. Organoids embedded within low stiffness gelatins (<2 kPa) are bioprinted into mathematically defined lattices with varying degrees of pore network tortuosity, and cultured under perfusion. These structures act as metabolic biofactories in which liver‐specific ammonia detoxification can be enhanced by the architectural profile of the constructs. This technology opens up new possibilities for regenerative medicine and personalized drug testing.

SummaryThe recent development of 3D-liver stem cell cultures (hepatic organoids) opens up new avenues for gene and/or stem cell therapy to treat liver disease. To test safety and efficacy, a relevant large animal model is essential but not yet established. Because of its shared pathologies and disease pathways, the dog is considered the best model for human liver disease. Here we report the establishment of a long-term canine hepatic organoid culture allowing undifferentiated expansion of progenitor cells that can be differentiated toward functional hepatocytes. We show that cultures can be initiated from fresh and frozen liver tissues using Tru-Cut or fine-needle biopsies. The use of Wnt agonists proved important for canine organoid proliferation and inhibition of differentiation. Finally, we demonstrate that successful gene supplementation in hepatic organoids of COMMD1-deficient dogs restores function and can be an effective means to cure copper storage disease.

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