Modeling PNPLA3 ‐Associated NAFLD Using Human‐Induced Pluripotent Stem Cells

Funder: NIH Oxford Cambridge Scholars ProgramFunder: Intramural Research Program of NIDDKFunder: European Research Council GrantFunder: Cambridge Hospitals National Institute for Health Research Biomedical Research CelnterFunder: Wellcome Trust and Medical Research Council Cambridge Stem Cell InsituteFunder: National Centre for the Replacement Refinement and Reduction of Animals in Research; Id: http://dx.doi.org/10.13039/501100010757Funder: Wellcome Sanger InstituteBackground and Aims: NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human‐induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology. Approach and Results: We used isogenic hiPSC lines with either a knockout (PNPLA3KO) of the PNPLA3 gene or with the I148M variant (PNPLA3I148M) to model PNPLA3‐associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD‐like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3KO hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid‐induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3KO cells to be more susceptible to ethanol‐induced and methotrexate‐induced toxicity. The PNPLA3I148M cells exhibited an intermediate phenotype between the wild‐type and PNPLA3KO cells. Conclusions: Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins

Transient treatment with ROCK inhibitors is sufficient to promote GSC-like cell expansion.

<p>The cells were treated with no inhibitor, with continuous exposure to the ROCK inhibitor (45 μM Y-27632 or 10 μM fasudil), or with transient exposure to the ROCK inhibitor (45 μM Y-27632 or 10 μM fasudil). (A) Representative micrographs of each experimental group on Day 3 (Scale bars = 100 μm). (B) The sphere diameter and number of spheres were analyzed for all experimental groups on Day 3 (mean ± SE; <i>n</i> = 100; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001). The number of spheres per field of view in each experimental group were also quantified from the micrographs (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

ROCK inhibitors enhance GSC-like stemness.

<p>(A) The clonogenicity of the cells was quantified using limiting dilution assay (steeper slope and lower value in x-intercept indicates increased clonogenic potential and stemness). The cells treated with 45 μM Y-27632 or 10 μM fasudil required fewer cells to form spheres indicating increased number of GSC-like cell than control. (B) The glioblastoma cells were grown as tumorspheres in two concentrations of Y-27632 (0 and 45μM) or two concentrations of fasudil (0 and 10 μM) for three days. Using flow cytometry, the percentage of the total population expressing the GSC marker SOX2 was quantified.</p

ROCK inhibitors are not toxic to GBM tumorspheres at low concentrations.

<p>The toxicities of Y-27632 and fasudil were measured using a water-soluble tetrazolium assay (WST-8 Cell Counting Kit 8). U87-MG, JX12, and SMC448 cells were exposed to varying concentrations of Y-27632 or fasudil for 48 hours. Cell viability was measured relative to 0 μM control (<i>n</i> = 10).</p

ROCK inhibitors protect GBM tumorspheres from apoptosis.

<p>Flow cytometry was used to quantify the late-stage apoptotic cells (Annexin V⁺/PI⁺) immediately after trituration. The cells that were treated with 45μM Y-27632 or 10 μM fasudil had decreased number of late-stage apoptotic cells in U87-MG, JX12, and SMC448 cell lines, indicating that the ROCK inhibitors Y-27632 and fasudil inhibited apoptosis in glioblastoma cells.</p

Knockdown of ROCK2 shows similar behavior to Y-27632 and Fasudil.

<p>U87-MG cells were transfected with ROCK2 siRNA and grown as tumorspheres for 3 days. The cells’ ability to form spheres was analyzed, and qRT-PCR was performed to confirm the success of the transfection. A) Representative micrographs of each experimental group on Day 1 (Scale bar = 100 μm). B) The sphere diameter (mean ± SE; <i>n</i> = 100; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001) and number of spheres per field of view were analyzed for all experimental groups on Day 1 (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001). C) qRT-PCR was performed on the transfected cells to confirm their gene expression levels of <i>ROCK2</i>, <i>CASP3</i>, and <i>CASP7</i>. Expression is reported as percentage of that of negative control (mean ± SE; <i>n</i> = 3; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

ROCK inhibitors enhance GBM tumorsphere formation.

<p>The glioblastoma cells were grown as tumorspheres in two concentrations of Y-27632 (0 and 45 μM) or two concentrations of fasudil (0 and 10μM) for 6 days. (A) Sample micrographs of each experimental group on Day 6 (Scale bars = 100 μm). (B) The sphere diameter and number of spheres were analyzed for all experimental groups on Day 3 (mean ± SE; <i>n</i> = 100). It was found that the sphere diameter stayed relatively consistent between the experimental groups. The number of spheres per field of view in each experimental group were also quantified from the micrographs (mean ± SE; <i>n</i> = 20; * <i>p</i> < 0.05, ** <i>p</i> < 0.01, and *** <i>p</i> < 0.001).</p

Regional Differences in Human Biliary Tissues and Corresponding In Vitro-Derived Organoids.

BACKGROUND AND AIMS: Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown. APPROACH AND RESULTS: Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions. CONCLUSIONS: Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development