Engineering a perfusable 3D human liver platform from iPS cells

© 2016 The Royal Society of Chemistry. In vitro models of human tissue are crucial to our ability to study human disease as well as develop safe and effective drug therapies. Models of single organs in static and microfluidic culture have been established and shown utility for modeling some aspects of health and disease; however, these systems lack multi-organ interactions that are critical to some aspects of drug metabolism and toxicity. Thus, as part of a consortium of researchers, we have developed a liver chip that meets the following criteria: (1) employs human iPS cells from a patient of interest, (2) cultures cells in perfusable 3D organoids, and (3) is robust to variations in perfusion rate so as to be compatible in series with other specialized tissue chips (e.g. heart, lung). In order to achieve this, we describe methods to form hepatocyte aggregates from primary and iPS-derived cells, alone and in co-culture with support cells. This necessitated a novel culture protocol for the interrupted differentiation of iPS cells that permits their removal from a plated surface and aggregation while maintaining phenotypic hepatic functions. In order to incorporate these 3D aggregates in a perfusable platform, we next encapsulated the cells in a PEG hydrogel to prevent aggregation and overgrowth once on chip. We adapted a C-trap chip architecture from the literature that enabled robust loading with encapsulated organoids and culture over a range of flow rates. Finally, we characterize the liver functions of this iHep organoid chip under perfusion and demonstrate a lifetime of at least 28 days. We envision that such this strategy can be generalized to other microfluidic tissue models and provides an opportunity to query patient-specific liver responses in vitro

Lgr5 intestinal stem cells have high telomerase activity and randomly segregate their chromosomes

This report reveals high telomerase activity and telomere length in dividing Lgr5-positive intestinal stem cells, features that decline during differentiation of stem cell progeny. Random segregation of chromosomes is observed, which is inconsistent with the “immortal strand” hypothesis

Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

The concept of 'field cancerization' describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5(+) stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations

Identification of NQO2 As a Protein Target in Small Molecule Modulation of Hepatocellular Function

The utility of in vitro human disease models is mainly dependent on the availability and functional maturity of tissue-specific cell types. We have previously screened for and identified small molecules that can enhance hepatocyte function in vitro. Here, we characterize the functional effects of one of the hits, FH1, on primary human hepatocytes in vitro, and also in vivo on primary hepatocytes in a zebrafish model. Furthermore, we conducted an analogue screen to establish the structure-activity relationship of FH1. We performed affinity-purification proteomics that identified NQO2 to be a potential binding target for this small molecule, revealing a possible link between inflammatory signaling and hepatocellular function in zebrafish and human hepatocyte model systems

The Cancer SENESCopedia: A delineation of cancer cell senescence

Cellular senescence is characterized as a stable proliferation arrest that can be triggered by multiple stresses. Most knowledge about senescent cells is obtained from studies in primary cells. However, senescence features may be different in cancer cells, since the pathways that are involved in senescence induction are often deregulated in cancer. We report here a comprehensive analysis of the transcriptome and senolytic responses in a panel of 13 cancer cell lines rendered senescent by two distinct compounds. We show that in cancer cells, the response to senolytic agents and the composition of the senescence-associated secretory phenotype are more influenced by the cell of origin than by the senescence trigger. Using machine learning, we establish the SENCAN gene expression classifier for the detection of senescence in cancer cell samples. The expression profiles and senescence classifier are available as an interactive online Cancer SENESCopedia.Pattern Recognition and Bioinformatic

Estrogen Activation of G-Protein–Coupled Estrogen Receptor 1 Regulates Phosphoinositide 3-Kinase and mTOR Signaling to Promote Liver Growth in Zebrafish and Proliferation of Human Hepatocytes

Background & Aims: Patients with cirrhosis are at high risk for hepatocellular carcinoma (HCC) and often have increased serum levels of estrogen. It is not clear how estrogen promotes hepatic growth. We investigated the effects of estrogen on hepatocyte proliferation during zebrafish development, liver regeneration, and carcinogenesis. We also studied human hepatocytes and liver tissues. Methods: Zebrafish were exposed to selective modifiers of estrogen signaling at larval and adult stages. Liver growth was assessed by gene expression, fluorescent imaging, and histologic analyses. We monitored liver regeneration after hepatocyte ablation and HCC development after administration of chemical carcinogens (dimethylbenzanthrazene). Proliferation of human hepatocytes was measured in a coculture system. We measured levels of G-protein–coupled estrogen receptor (GPER1) in HCC and nontumor liver tissues from 68 patients by immunohistochemistry. Results: Exposure to 17β-estradiol (E2) increased proliferation of hepatocytes and liver volume and mass in larval and adult zebrafish. Chemical genetic and epistasis experiments showed that GPER1 mediates the effects of E2 via the phosphoinositide 3-kinase–protein kinase B–mechanistic target of rapamycin pathway: gper1-knockout and mtor-knockout zebrafish did not increase liver growth in response to E2. HCC samples from patients had increased levels of GPER1 compared with nontumor tissue samples; estrogen promoted proliferation of human primary hepatocytes. Estrogen accelerated hepatocarcinogenesis specifically in male zebrafish. Chemical inhibition or genetic loss of GPER1 significantly reduced tumor development in the zebrafish. Conclusions: In an analysis of zebrafish and human liver cells and tissues, we found GPER1 to be a hepatic estrogen sensor that regulates liver growth during development, regeneration, and tumorigenesis. Inhibitors of GPER1 might be developed for liver cancer prevention or treatment. Transcript Profiling: The accession number in the Gene Expression Omnibus is GSE92544