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.

Stem cells are a unique source of self-renewing cells within the human body. Before the end of the last millennium, adult stem cells, in contrast to their embryonic counterparts, were considered to be lineage-restricted cells or incapable of crossing lineage boundaries. However, the unique breakthrough of muscle and liver regeneration by adult bone marrow stem cells at the end of the 1990s ended this long-standing paradigm. Since then, the number of articles reporting the existence of multipotent stem cells in skin, neuronal tissue, adipose tissue, and bone marrow has escalated, giving rise, both in vivo and in vitro, to cell types other than their tissue of origin. The phenomenon of fate reprogrammation and phenotypic diversification remains, though, an enigmatic and rare process. Understanding how to control both proliferation and differentiation of stem cells and their progeny is a challenge in many fields, going from preclinical drug discovery and development to clinical therapy. In this review, we focus on current strategies to differentiate embryonic, mesenchymal(-like), and liver stem/progenitor cells into hepatocytes in vitro. Special attention is paid to intracellular and extracellular signaling, genetic modification, and cell-cell and cell-matrix interactions. In addition, some recommendations are proposed to standardize, optimize, and enrich the in vitro production of hepatocyte-like cells out of stem/progenitor cells.

Background and Aims The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5)–positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Approach and Results Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40‐fold cell expansion after 2 weeks, compared with 6‐fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver‐like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up‐regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. Conclusions We established a highly efficient method for culturing large numbers of LGR5‐positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

SummarySo far, successful de novo formation of testicular tissue followed by complete spermatogenesis in vitro has been achieved only in rodents. Our findings reveal that primary human testicular cells are able to self-organize into human testicular organoids (TOs), i.e., multi-cellular tissue surrogates, either with or without support of a biological scaffold. Despite lacking testis-specific topography, these mini-tissues harbored spermatogonia and their important niche cells, which retained specific functionalities during long-term culture. These observations indicate the posibility of in vitro re-engineering of a human testicular microenvironment from primary cells. Human TOs might help in the development of a biomimetic testicular model that would exert a tremendous impact on research and development, clinical treatment of infertility, and screening in connection with drug discovery and toxicology.

Non-alcoholic fatty liver disease (NAFLD) affects one-third of the population worldwide, of which a substantial number of patients suffer from non-alcoholic steatohepatitis (NASH). NASH is a severe condition characterized by steatosis and concomitant liver inflammation and fibrosis, for which no drug is yet available. NAFLD is also generally conceived as the hepatic manifestation of the metabolic syndrome. Consequently, well-established drugs that are indicated for the treatment of type 2 diabetes and hyperlipidemia are thought to exert effects that alleviate the pathological features of NASH. One class of these drugs targets peroxisome proliferator-activated receptors (PPARs), which are nuclear receptors that play a regulatory role in lipid metabolism and inflammation. Therefore, PPARs are now also being investigated as potential anti-NASH druggable targets. In this paper, we review the mechanisms of action and physiological functions of PPARs and discuss the position of the different PPAR agonists in the therapeutic landscape of NASH. We particularly focus on the PPAR agonists currently under evaluation in clinical phase II and III trials. Preclinical strategies and how refinement and optimization may improve PPAR-targeted anti-NASH drug testing are also discussed. Finally, potential caveats related to PPAR agonism in anti-NASH therapy are stipulated.