Aras N. Mattis

Mouse liver repopulation with hepatocytes generated from human fibroblasts

Human induced pluripotent stem cells (iPSCs) have the capability of revolutionizing research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modelling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation. Here, as a solution to this problem, we report the generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs but cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this purpose we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of aHeps. Unfractionated iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state. Our results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.

Integration Host Factor: Putting a Twist on Protein–DNA Recognition

Integration host factor (IHF) is a DNA-bending protein that recognizes its cognate sites through indirect readout. Previous studies have shown that binding of wild-type (WT)-IHF is disrupted by a T to A mutation at the center position of a conserved TTR motif in its binding site, and that substitution of betaGlu44 with Ala prevented IHF from discriminating between A and T at this position. We have determined the crystal structures and relative binding affinities for all combinations of WT-IHF and IHF-betaGlu44Ala bound to the WT and mutant DNAs. Comparison of these structures reveals that DNA twist plays a major role in DNA recognition by IHF, and that this geometric parameter is dependent on the dinucleotide step and not on the bound IHF variant.

Clinical diagnosis of segmental arterial mediolysis: Differentiation from vasculitis and other mimics

Segmental arterial mediolysis (SAM) is a rare vasculopathy of unknown etiology characterized by disruption of the arterial medial layer, with resultant susceptibility to vessel dissection, hemorrhage, and ischemia. Since the first case of SAM described by Slavin and Gonzalez-Vitale in 1976 (1), approximately 50 cases have been reported in the literature (2). Although the abdominal visceral arteries are most frequently affected in SAM (3), any vessel may be involved, including the retroperitoneal (4), intracranial (2, 5, 6) and coronary arteries (7–9). The histopathologic changes begin with vacuolar degeneration of smooth muscle cells in the arterial media, followed by fibrin deposition at the medial-adventitial junction (7). This in turn predisposes to dissecting aneurysms (3, 10). The angiographic appearance of SAM is variable, ranging from arterial dilation to aneurysm formation (single or multiple) to stenoses or occlusion, frequently with dissection (3, 11). Correspondingly, symptoms arise both from stenoses and occlusions (e.g., postprandial pain from intestinal ischemia) and from dissections and aneurysms (e.g., sudden and catastrophic intraperitoneal bleeding). In contrast to true vasculitis, inflammatory cells in SAM are present inconsistently and, when present, are thought to be secondary rather than primary to the pathogenesis of the disease (1). The differential diagnosis of SAM includes atherosclerosis, fibromuscular dysplasia, infection (e.g., mycotic aneurysm and endocarditis), connective tissue diseases (e.g., Bechet’s disease and polyarteritis nodosa), neurofibromatosis, and inherited defects in vessel wall structural proteins (e.g., type IV Ehlers Danlos and Marfan’s syndrome) (Table 1). Herein, we describe two cases of SAM seen at our instituation over the past 20 years and review the salient clinical presentation and treatment of SAM. We report characteristics that may be helpful in distinguishing cases of SAM from other entities in the differential diagnosis. Table 1 Clinical and laboratory features distinguishing Segmental Arterial Mediolysis (SAM) from its mimics*. Case 1 The patient was a 25-year-old female who was admitted to the hospital with an eleven-month history of intermittent episodes of anorexia, abdominal pain, and diarrhea. Symptoms had persisted despite discontinuation of oral contraception and initiation of low-dose aspirin therapy. Her past medical history was unremarkable. Family history was unremarkable except for benign hypermobility syndrome in the patient’s mother. On physical examination, the patient was normotensive and had normal height and arm span. She had no carotid, subclavian, abdominal, or femoral bruits. Skin, chest, abdominal, and neurologic examinations were normal. Joint exam was remarkable only for hyperextensibility of the knees, reducible flexion contractures of the fingers, and hammertoe deformities of the feet. Complete blood count revealed anemia with hemoglobin of 11 g/dl. Serum creatinine, liver enzymes, amylase, and lipase were normal, and urine pregnancy test was negative. Antinuclear antibody assay (ANA) was positive to a low titer of (1:80). The erythrocyte sedimentation rate was 20 mm/hr. The remainder of the serologic, metabolic, immunologic, and hematologic evaluations were within normal limits, including negative hepatitis serologies, negative double-stranded DNA, anti-Smith, and anti-ribonucleoprotein (RNP) antibodies, and normal complement C3 and C4 levels. Computed tomography (CT) of the abdomen showed thickening of the colonic wall with mucosal enhancement and fat stranding surrounding the splenic flexure. Colonoscopy revealed ischemic colitis of the splenic flexure. Biopsies of the ischemic areas were not obtained due to risk of possible perforation. Biopsies of the non-ischemic areas were normal, as was magnetic resonance angiography (MRA) of the abdomen. Conventional mesenteric angiography revealed focal stenoses of the right and left hepatic arteries, occlusion of the left colic artery near the splenic flexure with collateral vessel formation, and hyperemia of multiple branches of the splenic artery (Figure 1). Figure 1 Angiographic and histologic features of segmental arterial mediolysis in Case 1. (a) Focal stenoses of the right and left hepatic arteries (arrows). (b) Hyperemic blush in the left colonic flexure, suggesting formation of small collateral vessels from ... Because of persistent ischemic colitis, the patient underwent a partial colectomy of the splenic flexure. Vascular pathology of the colonic arteries showed patchy, isolated destruction of the arterial media involving both the internal and external elastic laminae (Figure 1). In a few sections the media was absent, with direct juxtaposition of the intima and the adventitia. In areas of medial destruction, there was intimal proliferation with marked luminal narrowing. All of the lesions were of a similar age. There was no evidence of inflammation, and giant cells, neutrophils, and cholesterol deposits were absent. After two years of followup, the patient remains asymptomatic.

A screen in mice uncovers repression of lipoprotein lipase by microRNA‐29a as a mechanism for lipid distribution away from the liver

Identification of microRNAs (miRNAs) that regulate lipid metabolism is important to advance the understanding and treatment of some of the most common human diseases. In the liver, a few key miRNAs have been reported that regulate lipid metabolism, but since many genes contribute to hepatic lipid metabolism, we hypothesized that other such miRNAs exist. To identify genes repressed by miRNAs in mature hepatocytes in vivo, we injected adult mice carrying floxed Dicer1 alleles with an adenoassociated viral vector expressing Cre recombinase specifically in hepatocytes. By inactivating Dicer in adult quiescent hepatocytes we avoided the hepatocyte injury and regeneration observed in previous mouse models of global miRNA deficiency in hepatocytes. Next, we combined gene and miRNA expression profiling to identify candidate gene/miRNA interactions involved in hepatic lipid metabolism and validated their function in vivo using antisense oligonucleotides. A candidate gene that emerged from our screen was lipoprotein lipase (Lpl), which encodes an enzyme that facilitates cellular uptake of lipids from the circulation. Unlike in energy‐dependent cells like myocytes, LPL is normally repressed in adult hepatocytes. We identified miR‐29a as the miRNA responsible for repressing LPL in hepatocytes, and found that decreasing hepatic miR‐29a levels causes lipids to accumulate in mouse livers. Conclusion: Our screen suggests several new miRNAs are regulators of hepatic lipid metabolism. We show that one of these, miR‐29a, contributes to physiological lipid distribution away from the liver and protects hepatocytes from steatosis. Our results, together with miR‐29as known antifibrotic effect, suggest miR‐29a is a therapeutic target in fatty liver disease. (Hepatology 2015;61:141–152)

Human stem cells from single blastomeres reveal pathways of embryonic or trophoblast fate specification

Mechanisms of initial cell fate decisions differ among species. To gain insights into lineage allocation in humans, we derived ten human embryonic stem cell lines (designated UCSFB1-10) from single blastomeres of four 8-cell embryos and one 12-cell embryo from a single couple. Compared with numerous conventional lines from blastocysts, they had unique gene expression and DNA methylation patterns that were, in part, indicative of trophoblast competence. At a transcriptional level, UCSFB lines from different embryos were often more closely related than those from the same embryo. As predicted by the transcriptomic data, immunolocalization of EOMES, T brachyury, GDF15 and active β-catenin revealed differential expression among blastomeres of 8- to 10-cell human embryos. The UCSFB lines formed derivatives of the three germ layers and CDX2-positive progeny, from which we derived the first human trophoblast stem cell line. Our data suggest heterogeneity among early-stage blastomeres and that the UCSFB lines have unique properties, indicative of a more immature state than conventional lines. Highlighted article: Human ESCs derived from individual blastomeres of 8- and 12-cell human embryos have unique properties, provide insights into early human embryogenesis and enable human trophoblast stem cell derivation.

IntDOT Interactions with Core- and Arm-Type Sites of the Conjugative Transposon CTnDOT

CTnDOT is a Bacteroides conjugative transposon (CTn) that has facilitated the spread of antibiotic resistances among bacteria in the human gut in recent years. Although the integrase encoded by CTnDOT (IntDOT) carries the C-terminal set of conserved amino acids that is characteristic of the tyrosine family of recombinases, the reaction it catalyzes involves a novel step that creates a short region of heterology at the joined ends of the element during recombination. Also, in contrast to tyrosine recombinases, IntDOT catalyzes a reaction that is not site specific. To determine what types of contacts IntDOT makes with the DNA during excision and integration, we first developed an agarose gel-based assay for CTnDOT recombination, which facilitated the purification of the native IntDOT protein. The partially purified IntDOT was then used for DNase I footprinting analysis of the integration site attDOT and the excision sites attL and attR. Our results indicate that CTnDOT has five or six arm sites that are likely to be involved in forming higher-order nucleoprotein complexes necessary for synapsis. In addition, there are four core sites that flank the sites of strand exchange during recombination. Thus, despite the fact that the reaction catalyzed by IntDOT appears to be different from that typically catalyzed by tyrosine recombinases, the protein-DNA interactions required for higher-order structures and recombination appear to be similar.

Expression of glypican-3 in undifferentiated embryonal sarcoma and mesenchymal hamartoma of the liver.

Glypican-3 (GPC3) is an oncofetal protein that has been demonstrated to be a useful diagnostic immunomarker for hepatocellular carcinoma and hepatoblastoma. Its expression in mesenchymal tumors of the liver, particularly undifferentiated embryonal sarcoma (UES) and mesenchymal hamartoma (MH), has not been investigated. In this study, a total of 24 UESs and 18 MHs were immunohistochemically stained for GPC3 expression. The results showed cytoplasmic staining for GPC3 in 14 (58%) UESs, of which 6 exhibited diffuse immunoreactivity and the remaining 8 showed focal positivity. The patients with GPC3-positive UES tended to be younger (mean 18 years; median 11 years) than those with GPC3-negative tumors (mean 39.4 years; median 27 years), although the difference did not reach statistical significance (P = .06). Eight MHs also exhibited GPC3 immunoreactivity (44%; 4 diffuse and 4 focal). Positive staining in all 8 cases was primarily seen in entrapped nonlesional hepatocytes with a canalicular and cytoplasmic staining pattern. In only 4 cases (22%) was GPC3 immunoreactivity also observed in the mesenchymal component. The patients with positive staining also tended to be younger (mean 2.6 years; median 1.1 years) compared with those with negative staining (mean 16.3 years; median 4.5 years), but the difference was not statistically significant (P = .15). Our data demonstrate that GPC3 is expressed in a subset of UES and MH of the liver. Caution should thus be exercised when evaluating a GPC3-expressing hepatic neoplasm, particularly on a needle biopsy when the differential diagnosis includes poorly differentiated hepatocellular carcinoma or hepatoblastoma.

Purification and Characterization of Bacteriophage P22 Xis Protein

The temperate bacteriophages lambda and P22 share similarities in their site-specific recombination reactions. Both require phage-encoded integrase (Int) proteins for integrative recombination and excisionase (Xis) proteins for excision. These proteins bind to core-type, arm-type, and Xis binding sites to facilitate the reaction. lambda and P22 Xis proteins are both small proteins (lambda Xis, 72 amino acids; P22 Xis, 116 amino acids) and have basic isoelectric points (for P22 Xis, 9.42; for lambda Xis, 11.16). However, the P22 Xis and lambda Xis primary sequences lack significant similarity at the amino acid level, and the linear organizations of the P22 phage attachment site DNA-binding sites have differences that could be important in quaternary intasome structure. We purified P22 Xis and studied the protein in vitro by means of electrophoretic mobility shift assays and footprinting, cross-linking, gel filtration stoichiometry, and DNA bending assays. We identified one protected site that is bent approximately 137 degrees when bound by P22 Xis. The protein binds cooperatively and at high protein concentrations protects secondary sites that may be important for function. Finally, we aligned the attP arms containing the major Xis binding sites from bacteriophages lambda, P22, L5, HP1, and P2 and the conjugative transposon Tn916. The similarity in alignments among the sites suggests that Xis-containing bacteriophage arms may form similar structures.

De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation

Transdifferentiation is a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, in the presence of a fully developed scaffold and niche1. Here we show that transdifferentiation of hepatocytes in the mouse liver can build a structure that failed to form in development—the biliary system in a mouse model that mimics the hepatic phenotype of human Alagille syndrome (ALGS)2. In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, that is, incomplete and transient biliary differentiation as an adaptation to cell injury, based on previous studies in mice with a fully developed biliary system3–6. In contrast to bile duct development7–9, we show that de novo bile duct formation by hepatocyte transdifferentiation is independent of NOTCH signalling. We identify TGFβ signalling as the driver of this compensatory mechanism and show that it is active in some patients with ALGS. Furthermore, we show that TGFβ signalling can be targeted to enhance the formation of the biliary system from hepatocytes, and that the transdifferentiation-inducing signals and remodelling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. Our results define the regenerative potential of mammalian transdifferentiation and reveal opportunities for the treatment of ALGS and other cholestatic liver diseases.In a mouse model of a human cholestatic liver disease caused by impaired NOTCH signalling, hepatocytes transdifferentiate into cholangiocytes and form a therapeutically effective biliary system, driven by TGFβ signalling.

Association of Aneuploidy and Flat Dysplasia With Development of High-Grade Dysplasia or Colorectal Cancer in Patients With Inflammatory Bowel Disease

There is controversy over how to best manage patients with inflammatory bowel disease and flat low-grade dysplasia (fLGD) in the colon. We performed a retrospective analysis of formalin-fixed paraffin-embedded colon tissues with fLGD from 37 patients undergoing surveillance colonoscopy for inflammatory bowel disease from 1990 to 2015 at the University of California at San Francisco Medical Center, to determine whether detection of aneuploidy is associated with later development of high-grade dysplasia (HGD) or colorectal cancer. Medical data were collected from the patients for a mean follow-up time of 37 months. Using flow cytometry analysis of paraffin-embedded colon tissue, we detected aneuploidy in 15 of 37 samples with fLGD (40.5%). By comparison, aneuploidy was detected in 14 of 15 samples with flat HGD (93.3%) and 2 of 45 samples that were negative for dysplasia (4.4%). The univariate hazard ratio for subsequent detection of HGD or colorectal cancer in patients with fLGD and aneuploidy was 5.3 (95% CI, 1.542-24.121) within a mean follow-up time of 37 months. The presence of aneuploidy therefore identifies patients with fLGD in colon tissue who have an increased risk for HGD or colorectal cancer and may provide supportive evidence to a morphologic impression or suspicion of flat HGD.