Previous research has indicated that liver androgen receptors may play a role in modulating disease. This study aims to investigate the pathophysiology of high-fat diet (HFD) induced dysglycemia in male and female liver androgen receptor knockout (LivARKO) mice. We performed metabolic tests on LivARKO female and male mice fed a HFD or a control diet (from Research Diets Inc.) during months 1 or 2 after starting the diet. Additionally, we performed Western blot and quantitative real-time PCR analysis on the livers of the mice to examine intermediates in the insulin signaling pathway. LivARKO-HFD female mice displayed no difference in glucose tolerance compared to female LivARKO-Control (Con) mice, whereas in wild-type female mice, HFD impaired glucose tolerance (IGT). Our data suggests that starting at 1 month, LivARKO may be protecting female mice from HFD-induced metabolic dysfunction. LivARKO-HFD female mice displayed significantly worse insulin sensitivity at 15 minutes compared to LivARKO-Con female mice, but, strangely, LivARKO-HFD female mice had significantly better insulin sensitivity at 60 and 90 minutes compared to LivARKO-Con female mice. Despite protecting against IGT, LivARKO did not protect against HFD-induced hyperinsulinemia in female mice. In contrast to females, male LivARKO-HFD mice displayed impaired glucose tolerance compared to male LivARKO-Con mice. Thus, LivARKO is not protective against HFD-induced glucose metabolic dysfunction in male mice. Lastly, LivARKO-HFD female mice maintained hepatic insulin sensitivity whereas LivARKO-HFD male mice displayed hepatic insulin resistance. These findings suggest that LivARKO delayed the onset of HFD-induced dysglycemia in female mice.
Somatic Liver Knockout (SLiK): A Quick and Efficient Way to Generate Liver‐Specific Knockout Mice Using Multiplex CRISPR/Cas9 Gene Editing
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Abstract Human induced pluripotent stem cell (iPSC)-derived liver organoids serve as models of organogenesis, disease, drug screening, and regenerative medicine. Prevailing methods for generating organoids rely on Matrigel, whose batch-to-batch variability and xenogeneic source pose challenges to mechanistic research and translation to human clinical therapy. In this report, we demonstrate that self-assembled Matrigel-free iPSC-derived organoids developed in rotating wall vessels (RWVs) exhibit greater hepatocyte-specific functions than organoids formed on Matrigel. We show that RWVs produce highly functional liver organoids in part by eliminating the need for Matrigel, which has adverse effects on hepatic lineage differentiation. RWV liver organoids sustain durable function over long-term culture and express a range of mature functional genes at levels comparable to adult human liver, while retaining some fetal features. Our results indicate that RWVs provide a simple and high-throughput way to generate Matrigel-free liver organoids suitable for research and clinical applications.more » « less
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2938 Using a Human Liver Tissue Equivalent (hLTE) Platform to Define the Functional Impact of Liver-Directed AAV Gene Therapy 63rd ASH Annual Meeting and Exposition, December 11-14, 2021, Georgia World Congress Center, Atlanta, GA Program: Oral and Poster Abstracts Session: 801. Gene Therapies: Poster II Hematology Disease Topics & Pathways: Bleeding and Clotting, Biological, Translational Research, Hemophilia, Genetic Disorders, Clinically Relevant, Diseases, Gene Therapy, Therapies Sunday, December 12, 2021, 6:00 PM-8:00 PM Ritu M Ramamurthy1*, Wen Ting Zheng2*, Sunil George, PhD1*, Meimei Wan1*, Yu Zhou, PhD1*, Baisong Lu, PhD1*, Colin E Bishop, PhD1*, Anthony Atala, M.D.1*, Christopher D Porada, PhD1* and M. Graca Almeida-Porada, MD3 1Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC 2Massachusetts Institute of Technology, Cambridge, MA 3Fetal Research and Therapy Program, Wake Forest Institute For Regenerative Medicine, Winston-Salem, NC Clinical trials employing AAV vectors for hemophilia A have been hindered by unanticipated immunological and/or inflammatory responses in some of the patients. Also, these trials have often yielded lower levels of transgene expression than were expected based upon preclinical studies, highlighting the poor correlation between the transduction efficiency observed in traditional 2D cultures of primary cells in vitro, and that observed in those same cell types in vivo. It has been also recognized that there are marked species-specific differences in AAV-vector tropism, raising the critical question of the accuracy with which various animal models will likely predict tropism/vector transduction efficiency, and eventual treatment success in humans. Human liver tissue equivalents (hLTEs) are comprised of major cell types in the liver in physiologically relevant frequencies and possess the ability to recapitulate the biology and function of native human liver. Here, we hypothesize that hLTEs can be used as a better model to predict the efficacy and safety of AAV gene therapy in humans. We fabricated hLTEs using 75% hepatocytes, 10% stellate cells, 10% Kupffer cells, and 5% liver sinusoid-derived endothelial cells in 96-well Elplasia plates with 79 microwells per well. hLTEs were transduced at an MOI of 105vg/cell, on the day of fabrication, with the clinically relevant serotypes AAV5 (hLTE-5) or AAV3b (hLTE-3b), both encoding a GFP reporter. After 4 days of self-aggregation, live/dead assay was performed to confirm viability. Non-transduced hLTEs served as negative controls (hLTE(-)), and hLTEs exposed to 20 mM acetaminophen were used as positive controls for liver inflammation/damage. Incucyte® Live-Cell Imaging system was used to track the aggregation and GFP expression of hLTEs. Over the course of the next 5 days, media was collected to determine hepatic functionality, RNA was isolated to assess dysregulation of genes involved in inflammation and fibrosis, DNA was isolated to determine whether AAV vectors integrate into the genome of human hepatocytes and, if so, to define the frequency at which this occurs and the genomic loci of integration, and hLTEs were fixed and processed at appropriate times for histological analyses and transmission electron microscopy (TEM). TEM analysis revealed that all groups exhibited microvilli and bile-canaliculus-like structures, demonstrating the formation of a rudimentary biliary system and, more importantly, proving that hLTEs resemble native liver structure. Incucyte® imaging showed that AAV5 and AAV3b transduction impaired formation of hLTEs (57.57 ± 2.42 and 24.57 ± 4.01 spheroids/well, respectively) in comparison with hLTE(-) (74.86 ± 3.8 spheroids/well). Quantification of GFP expression demonstrated that AAV5 yielded the most efficient transduction of hLTEs (fold change in GFP expression compared to control: 2.73 ± 0.09 and 1.19 ± 0.03 for hLTE-5 and hLTE-3b, respectively). Chromogenic assays showed decreased urea production in cell culture supernatants of AAV transduced groups compared to the non-transduced hLTEs on days 6 and 10 of culture, demonstrating decreased hepatocyte functionality. However, ALT and AST levels were similar in all groups. On day 10, hLTEs were either used for RNA isolation or fixed in 4% PFA and processed for histology. Masson’s Trichrome and Alcian Blue/Sirius Red staining was performed to detect fibrosis, which was then quantified using ImageJ. These analyses showed no significant increase in fibrosis in either hLTE-5 or hLTE-3b compared to hLTE(-). Nevertheless, RT2 PCR Array for Human Fibrosis detected dysregulation of several genes involved in fibrosis/inflammation in both hLTE-5 and hLTE-3b (16/84 and 26/84, respectively). In conclusion, data collected thus far show successful recapitulation of native liver biology and demonstrate that AAV5 transduces hLTEs more efficiently than AAV3b. However, impaired self-aggregation and decreased hepatocyte functionality was observed in both AAV-transduced groups. Studies to address the incidence and location(s) of AAV integration are ongoing. We have thus shown that the hLTE system can provide critical new knowledge regarding the efficacy and safety of AAV gene therapy in the human liver. Disclosures: No relevant conflicts of interest to declare.more » « less
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Drug-induced liver injury (DILI) remains a leading cause of drug attrition and acute liver failures, partly due to the inadequacy of animal models to accurately predict human clinical outcomes, which necessitates the utilization of in vitro models of the human liver. However, primary human hepatocytes (PHHs) are in short supply for routine drug screening. In contrast, induced pluripotent stem cells (iPSCs)-derived hepatocyte-like cells (HLCs) are a nearly unlimited cell source but display a fetal-like (versus adult-like) phenotype when differentiated using conventional protocols on tissue culture plastic or glass adsorbed with 2D extracellular matrix (ECM) proteins. Electrospinning can produce porous nanoscale 3D fibers that have a large surface area and present a high density of receptor ligands to modulate cell phenotype. However, the application of electrospinning to generate 3D liver-derived ECM substrates for HLC differentiation remains unexplored. Therefore, here we developed methods to a) electrospin nanofibers of different porosities and diameters using porcine liver ECM (PLECM) with or without type I collagen and b) use these fibers to determine functional modulation in iPSC-derived HLCs while using PHHs as a control cell type relative to conventional adsorbed ECM substrates.more » « less
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Abstract The liver possesses a unique microenvironment with a complex internal vascular system and cell–cell interactions. Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, and although much effort has been dedicated to building models to target NAFLD, most in vitro systems rely on simple models failing to recapitulate complex liver functions. Here, an in vitro system is presented to study NAFLD (steatosis) by coculturing human hepatocellular carcinoma (HepG2) cells and umbilical vein endothelial cells (HUVECs) into spheroids. Analysis of colocalization of HepG2–HUVECs along with the level of steatosis reveals that the NAFLD pathogenesis could be better modeled when 20% of HUVECs are presented in HepG2 spheroids. Spheroids with fat supplements progressed to the steatosis stage on day 2, which could be maintained for more than a week without being harmful for cells. Transferring spheroids onto a chip system with an array of interconnected hexagonal microwells proves helpful for monitoring functionality through increased albumin secretions with HepG2–HUVEC interactions and elevated production of reactive oxygen species for steatotic spheroids. The reversibility of steatosis is demonstrated by simply stopping fat‐based diet or by antisteatotic drug administration, the latter showing a faster return of intracellular lipid levels to the basal level.
