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. 2018 Oct;69(4):851-860.
doi: 10.1016/j.jhep.2018.05.028. Epub 2018 Jun 5.

hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency

Charis-Patricia Segeritz  1 , Sheikh Tamir Rashid  2 , Miguel Cardoso de Brito  3 , Maria Paola Serra  4 , Adriana Ordonez  5 , Carola Maria Morell  3 , Joseph E Kaserman  6 , Pedro Madrigal  3 , Nicholas R F Hannan  3 , Laurent Gatto  7 , Lu Tan  5 , Andrew A Wilson  6 , Kathryn Lilley  7 , Stefan J Marciniak  5 , Bibek Gooptu  8 , David A Lomas  9 , Ludovic Vallier  10
Affiliations

hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency

Charis-Patricia Segeritz et al. J Hepatol. 2018 Oct.

Abstract

Background & aims: α1-Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies.

Methods: Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and "omics" comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures.

Results: Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line.

Conclusions: Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD.

Lay summary: This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.

Keywords: Hepatocyte; Human-induced pluripotent stem cell; Inflammation; Inherited liver disease; α(1)-Antitrypsin deficiency.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Validating a cellular platform suitable for in-depth disease characterisation. (A) qPCR analyses for the denoted genes in ZZ- (mutant, n = 12) and RR- (corrected, n = 12) HLCs vs. positive controls (plated and fresh, cryopreserved human primary hepatocytes, n = 2–3). (B) DELFIA of secreted α1-antitrypsin and albumin in ZZ- and RR-HLCs; n = 3–9. (C) Immunostaining of ZZ and RR-HLCs for HNFα, α1-antitrypsin or albumin. Scale bar: 100 μm. (D) Pulse-chase analysis: Radioactively labelled, newly-synthesised protein was tracked from the intracellular (lysate) to extracellular space (supernatant) at 1 h, 2 h and 4 h. HLCs, hepatocyte-like cells; qPCR, quantitative PCR. Statistical analyses of (A) and (B) by unpaired, parametric t test: n.s. (non-significant), *p <0.05, **p ≤0.01, ***p <0.001, ****p ≤0.0001.
Fig. 2
Fig. 2
ZZ-HLCs display heterogenous abnormal ER morphology. (A) ER marker GFP-KDEL shows the different types of abnormal ER morphologies observed in ZZ-HLCs vs. RR-HLCs. (B) Quantification of each ER morphology. ER, endoplasmic reticulum; GFP, green fluorescent protein; HLCs, hepatocyte-like cells.
Fig. 3
Fig. 3
Fluorescence loss in photobleaching shows absence of intracellular exchanges between ER inclusions. (A) FLIP of ZZ- and RR-HLCs transfected with KDEL-GFP: Pre-bleach fluorescence was determined before bleaching. Fluorescence was monitored within equidistant areas (stars) from the area of bleach (red box). Graphs show normalised FLIP plots with pre-bleach intensities set to 100%. n = 5. (B) FLIP of ZZ-HLCs with large inclusions transfected with CytERM. Graph shows normalised FLIP plot with pre-bleach intensities set to 100%. n = 3. (C) 100 μm Z-tack image of ZZ-HLCs. Scale bars: 10 μm. FLIP, fluorescence loss in photobleaching; GFP, green fluorescent protein; HLCs, hepatocyte-like cells.
Fig. 4
Fig. 4
Fluorescent recovery after photobleaching confirming decreased ER protein mobility in ZZ-HLCs. (A) KDEL-GFP transfected cells were exposed to a single bleaching event (white box). (B) Recovery of GFP fluorescence by way of diffusion into the bleached area was measured by tracking fluorescence within the bleached area. (C) Slope of the fluorescent recovery curve and the mobile fraction indicate a significantly lower degree of protein mobility and higher viscosity in ZZ-HLCs with inclusions. Statistical analysis by unpaired, parametric t test: **p ≤0.01, ****p ≤0.0001. ER, endoplasmic reticulum; FRAP, fluorescent recovery after photobleaching; GFP, green fluorescent protein; HLCs, hepatocyte-like cells.
Fig. 5
Fig. 5
Proteomic and RNA-seq analyses capture novel disease signatures. (A) Experimental process for proteomic analyses. (B) List of proteins identified in the ER proteome (green: present) and (red: absent) between ZZ- and RR-HLC. (C) Microarray plot shows log2 fold changes in gene expression between ZZ- and RR-HLCs over the mean of normalised counts. Significant genes with Benjamini-Hochberg FDR < 0.05 are shown in red. (D) 319 gene-protein pairs with differential abundances and gene expression were identified (blue, adjusted p values >0.05 in red). (E) Most differentially expressed gene/protein pairs upregulated (top) or downregulated (bottom). Horizontal lines: parameter boundaries of log2-fold change thresholds >2 and <−2. (F) Data clustering showed the FPKM-based genes upregulated in ZZ-HLCs mapped into four groups: very high, high, low or very low expression. (G) Enriched gene ontology terms (p value <0.05) obtained from BioMart for the analysis of genes upregulated in ZZ-HLCs. ER, endoplasmic reticulum; FDR, false discovery rate; FPKM, fragments per kilobase of transcript per million mapped reads; HLCs, hepatocyte-like cells.
Fig. 6
Fig. 6
Clinical relevance of novel disease signatures. (A) Immunostaining of PiZZ patient liver tissue sections confirmed (green arrow) and absence (red arrow) of PAS-positive α1-antitrypsin globules (counter stained with polymer specific 2C1 antibody). Scale bar: 10 μm. (B) 1,675 differentially regulated genes identified by RNA-seq of ZZ vs. RR-HLCs were compared to the top 1,675 genes with the lowest p values obtained by RNA-seq of one PiZZ vs. PiMM primary tissue sample. 80 genes were shared between the two gene lists. (C) AKR1B10 immunostaining of liver sections (n = 3 patients) with clinical backgrounds matching those of the original hIPSC donor. Staining with the 2C1 antibody confirmed presence of polymer in same cells as AKR1B10; negative controls (secondary antibody alone). Scale bar: 20 μm. (D) Electron microscopy comparing mitochondria in ZZ vs. RR-HLCs. Organelles with highly compressed morphology yielding heterogeneous electron densities are indicated (red arrows). Scale bar: 2 μm. hiPSC, human-induced pluripotent stem cells; HLCs, hepatocyte-like cells.
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