Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by inheritance of the SERPINA1 'Z' genetic variant (PiZ) driving AAT protein misfolding in hepatocytes. There remain no approved medicines for this disease. Here, we report the results of a small molecule screen performed in patient derived iPSC-hepatocytes that identified Leucine-rich repeat kinase-2 (LRRK2) as a potentially new therapeutic target. Of the commercially available LRRK2 inhibitors tested, we identified CZC-25146, a candidate with favorable pharmacokinetic properties, as being capable of reducing polymer load, increasing normal AAT secretion, and reducing inflammatory cytokines in both cells and PiZ mice. Mechanistically, this effect was achieved through induction of autophagy. Our findings support the use of CZC-25146 and LRRK2 inhibitors in hepatic proteinopathy research and their further investigation as novel therapeutic candidates for A1ATD.

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Structural basis for the modulation of MRP2 activity by phosphorylation and drugs.

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

Induced pluripotent stem cells: from Nobel Prizes to clinical applications.

Advances in basic hepatology have been constrained for many years by the inability to culture primary hepatocytes in vitro, until just over five years ago when the scientific playing field was changed beyond recognition with the demonstration that human skin fibroblasts could be reprogrammed to resemble embryonic cells. The reprogrammed cells, known as induced pluripotent stem cells (iPSCs), were then shown to have the capacity to re-differentiate into almost any human cell type, including hepatocytes. The unlimited number and isogenic nature of the cells that can be generated from tiny fragments of tissue have massive implications for the study of human liver diseases in vitro. Of more immediate clinical importance were recent data demonstrating precision gene therapy on patient specific iPSCs, which opens up the real and exciting possibility of autologous hepatocyte transplantation as a substitute for allogeneic whole liver transplantation, which has been an effective approach to end-stage liver disease, but one that has now been outstripped by demand. In this review, we describe the historical development, current technology and potential clinical applications of induced pluripotency, concluding with a perspective on possible future directions in this dynamic field.

RGD density along with substrate stiffness regulate hPSC hepatocyte functionality through YAP signalling.

Human pluripotent stem cell-derived hepatocytes (hPSC-Heps) may be suitable for treating liver diseases, but differentiation protocols often fail to yield adult-like cells. We hypothesised that replicating healthy liver niche biochemical and biophysical cues would produce hepatocytes with desired metabolic functionality. Using 2D synthetic hydrogels which independently control mechanical properties and biochemical cues, we found that culturing hPSC-Heps on surfaces matching the stiffness of fibrotic liver tissue upregulated expression of genes for RGD-binding integrins, and increased expression of YAP/TAZ and their transcriptional targets. Alternatively, culture on soft, healthy liver-like substrates drove increases in cytochrome p450 activity and ureagenesis. Knockdown of ITGB1 or reducing RGD-motif-containing peptide concentration in stiff hydrogels reduced YAP activity and improved metabolic functionality; however, on soft substrates, reducing RGD concentration had the opposite effect. Furthermore, targeting YAP activity with verteporfin or forskolin increased cytochrome p450 activity, with forskolin dramatically enhancing urea synthesis. hPSC-Heps could also be successfully encapsulated within RGD peptide-containing hydrogels without negatively impacting hepatic functionality, and compared to 2D cultures, 3D cultured hPSC-Heps secreted significantly less fetal liver-associated alpha-fetoprotein, suggesting furthered differentiation. Our platform overcomes technical hurdles in replicating the liver niche, and allowed us to identify a role for YAP/TAZ-mediated mechanosensing in hPSC-Hep differentiation.

Proteomic analysis of extracellular matrix from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a as novel constituents of fibrotic liver.

Activation of hepatic stellate cells (HSCs) and subsequent uncontrolled accumulation of altered extracellular matrix (ECM) underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. We sought to catalogue the components of fibrotic liver ECM to obtain insights into disease etiology and aid identification of new biomarkers. Cell-derived ECM was isolated from the HSC line LX-2, an in vitro model of liver fibrosis, and compared to ECM from human foreskin fibroblasts (HFFs) as a control. Mass spectrometry analyses of cell-derived ECMs identified, with ≥99% confidence, 61 structural ECM or secreted proteins (48 and 31 proteins for LX-2 and HFF, respectively). Gene ontology enrichment analysis confirmed the enrichment of ECM proteins, and hierarchical clustering coupled with protein-protein interaction network analysis revealed a subset of proteins enriched to fibrotic ECM, highlighting the existence of cell type-specific ECM niches. Thirty-six proteins were enriched to LX-2 ECM as compared to HFF ECM, of which Wnt-5a and CYR61 were validated by immunohistochemistry in human and murine fibrotic liver tissue. Future studies will determine if these and other components may play a role in the etiology of hepatic fibrosis, serve as novel disease biomarkers, or open up new avenues for drug discovery.

Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.

Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.

Sulfated Alginate Reduces Pericapsular Fibrotic Overgrowth on Encapsulated cGMP-Compliant hPSC-Hepatocytes in Mice.

Intra-peritoneal placement of alginate encapsulated human induced pluripotent stem cell-derived hepatocytes (hPSC-Heps) represents a potential new bridging therapy for acute liver failure. One of the rate-limiting steps that needs to be overcome to make such a procedure more efficacious and safer is to reduce the accumulation of fibrotic tissue around the encapsulated cells to allow the free passage of relevant molecules in and out for metabolism. Novel chemical compositions of alginate afford the possibility of achieving this aim. We accordingly used sulfated alginate and demonstrated that this material reduced fibrotic overgrowth whilst not impeding the process of encapsulation nor cell function. Cumulatively, this suggests sulfated alginate could be a more suitable material to encapsulate hPSC-hepatocyte prior to human use.

Induced pluripotent stem cells--alchemist's tale or clinical reality?

Following Shinya Yamanaka's first report describing the reprogramming of fibroblasts into stem cells over three years ago, some sceptics initially drew analogies between this new field of research and the quasi-mystical practice of 'alchemy'. Unlike the alchemist, however, stem cell researchers have rigorously tested and repeated experiments, proving their very own brand of cellular 'alchemy' to be a reality, with potentially massive implications for the study of human biology and clinical medicine. These investigations have resulted in an explosion of related publications and initiated the field of stem cell research known as 'induced pluripotency'. In this review, we give an account of the historical development, current technologies and potential clinical applications of induced pluripotency and conclude with a perspective on the possible future directions for this dynamic field.

The structural basis for Z α1-antitrypsin polymerization in the liver.

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α1-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α1-antitrypsin as "polymer" chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α1-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α1-antitrypsin.

Validation of Current Good Manufacturing Practice Compliant Human Pluripotent Stem Cell-Derived Hepatocytes for Cell-Based Therapy.

Recent advancements in the production of hepatocytes from human pluripotent stem cells (hPSC-Heps) afford tremendous possibilities for treatment of patients with liver disease. Validated current good manufacturing practice (cGMP) lines are an essential prerequisite for such applications but have only recently been established. Whether such cGMP lines are capable of hepatic differentiation is not known. To address this knowledge gap, we examined the proficiency of three recently derived cGMP lines (two hiPSC and one hESC) to differentiate into hepatocytes and their suitability for therapy. hPSC-Heps generated using a chemically defined four-step hepatic differentiation protocol uniformly demonstrated highly reproducible phenotypes and functionality. Seeding into a 3D poly(ethylene glycol)-diacrylate fabricated inverted colloid crystal scaffold converted these immature progenitors into more advanced hepatic tissue structures. Hepatic constructs could also be successfully encapsulated into the immune-privileged material alginate and remained viable as well as functional upon transplantation into immune competent mice. This is the first report we are aware of demonstrating cGMP-compliant hPSCs can generate cells with advanced hepatic function potentially suitable for future therapeutic applications. Stem Cells Translational Medicine 2019;8:124&14.

Single cell analysis of human foetal liver captures the transcriptional profile of hepatobiliary hybrid progenitors.

The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM+ population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.

Imaging-Based Screen Identifies Laminin 411 as a Physiologically Relevant Niche Factor with Importance for i-Hep Applications.

Use of hepatocytes derived from induced pluripotent stem cells (i-Heps) is limited by their functional differences in comparison with primary cells. Extracellular niche factors likely play a critical role in bridging this gap. Using image-based characterization (high content analysis; HCA) of freshly isolated hepatocytes from 17 human donors, we devised and validated an algorithm (Hepatocyte Likeness Index; HLI) for comparing the hepatic properties of cells against a physiological gold standard. The HLI was then applied in a targeted screen of extracellular niche factors to identify substrates driving i-Heps closer to the standard. Laminin 411, the top hit, was validated in two additional induced pluripotent stem cell (iPSC) lines, primary tissue, and an in vitro model of α1-antitrypsin deficiency. Cumulatively, these data provide a reference method to control and screen for i-Hep differentiation, identify Laminin 411 as a key niche protein, and underscore the importance of combining substrates, soluble factors, and HCA when developing iPSC applications.

Human iPS derived progenitors bioengineered into liver organoids using an inverted colloidal crystal poly (ethylene glycol) scaffold.

Generation of human organoids from induced pluripotent stem cells (iPSCs) offers exciting possibilities for developmental biology, disease modelling and cell therapy. Significant advances towards those goals have been hampered by dependence on animal derived matrices (e.g. Matrigel), immortalized cell lines and resultant structures that are difficult to control or scale. To address these challenges, we aimed to develop a fully defined liver organoid platform using inverted colloid crystal (ICC) whose 3-dimensional mechanical properties could be engineered to recapitulate the extracellular niche sensed by hepatic progenitors during human development. iPSC derived hepatic progenitors (IH) formed organoids most optimally in ICC scaffolds constructed with 140 µm diameter pores coated with type I collagen in a two-step process mimicking liver bud formation. The resultant organoids were closer to adult tissue, compared to 2D and 3D controls, with respect to morphology, gene expression, protein secretion, drug metabolism and viral infection and could integrate, vascularise and function following implantation into livers of immune-deficient mice. Preliminary interrogation of the underpinning mechanisms highlighted the importance of TGFß and hedgehog signalling pathways. The combination of functional relevance with tuneable mechanical properties leads us to propose this bioengineered platform to be ideally suited for a range of future mechanistic and clinical organoid related applications.

Stem cell-based therapy for α1-antitrypsin deficiency.

Human induced pluripotent stem cells offer the possibility of generating unlimited quantities of cells for autologous transplantation. By correcting the genetic defect underlying Z-allele α1-antitrypsin deficiency, we recently provided the first proof of principle for application of human induced pluripotent stem cells in the treatment of inherited genetic disorders. Several important safety concerns will need to be addressed before this can be translated into clinical practice.

The serpinopathies studying serpin polymerization in vivo.

The serpinopathies result from point mutations in members of the serine protease inhibitor or serpin superfamily. They are characterized by the formation of ordered polymers that are retained within the cell of synthesis. This causes disease by a "toxic gain of function" from the accumulated protein and a "loss of function" as a result of the deficiency of inhibitors that control important proteolytic cascades. The serpinopathies are exemplified by the Z (Glu342Lys) mutant of α1-antitrypsin that results in the retention of ordered polymers within the endoplasmic reticulum of hepatocytes. These polymers form the intracellular inclusions that are associated with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. A second example results from mutations in the neurone-specific serpin-neuroserpin to form ordered polymers that are retained as inclusions within subcortical neurones as Collins' bodies. These inclusions underlie the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. There are different pathways to polymer formation in vitro but not all form polymers that are relevant in vivo. It is therefore essential that protein-based structural studies are interpreted in the context of human samples and cell and animal models of disease. We describe here the biochemical techniques, monoclonal antibodies, cell biology, animal models, and stem cell technology that are useful to characterize the serpin polymers that form in vivo.

Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells.

Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (alpha1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded alpha1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor-mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.