Potential of human induced pluripotent stem cells in studies of liver disease.

Liver disease is a leading cause of death in the Western world. However, our insight into the underlying disease mechanisms and the development of novel therapeutic agents has been hindered by limited availability of primary tissue, intraspecies variability associated with the use of animal models, and reduced long-term viability of isolated and diseased liver cells. The emergence of human induced pluripotent stem cells and differentiation protocols to generate hepatocyte-like cells has opened the possibility of addressing these issues. Here, we discuss the recent progress and potential in the production of various cell types constituting the liver and their applications to model liver diseases and test drug toxicity in vitro.FS is supported by an Addenbrooke’s Charitable Trust Clinical Research Training Fellowship, a joint Sparks-MRC Clinical Research Training Fellowship and the Cambridge Hospitals National Institute for Health Research Biomedical Research Center. CPS is supported by the Children’s Liver Disease Foundation. LV is supported by the ERC starting grant Relieve IMDs, the Cambridge Hospitals National Institute for Health Research Biomedical Research Center and the EuFp7 grants InnovaLIV and TissuGEN.This is the accepted manuscript. The final published version is available from Wiley at http://onlinelibrary.wiley.com/doi/10.1002/hep.27651/abstract

A proteomic time course through the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

Numerous in vitro models endeavour to mimic the characteristics of primary human hepatocytes for applications in regenerative medicine and pharmaceutical science. Mature hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSCs) are one such in vitro model. Due to insufficiencies in transcriptome to proteome correlation, characterising the proteome of HLCs is essential to provide a suitable framework for their continual optimization. Here we interrogated the proteome during stepwise differentiation of hiPSCs into HLCs over 40 days. Whole cell protein lysates were collected and analysed using stabled isotope labelled mass spectrometry based proteomics. Quantitative proteomics identified over 6,000 proteins in duplicate multiplexed labelling experiments across two different time course series. Inductive cues in differentiation promoted sequential acquisition of hepatocyte specific markers. Analysis of proteins classically assigned as hepatic markers demonstrated trends towards maximum relative abundance between differentiation day 30 and 32. Characterisation of abundant proteins in whole cells provided evidence of the time dependent transition towards proteins corresponding with the functional repertoire of the liver. This data highlights how far the proteome of undifferentiated precursors have progressed to acquire a hepatic phenotype and constructs a platform for optimisation and improved maturation of HLC differentiation

Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.This work was funded by the ERC starting grant Relieve IMDs (L.V.), the Cambridge Hospitals National Institute for Health Research Biomedical Research Center (L.V., N.R.F.H.), and the Evelyn trust (N.R.F.H.). N.A.H. is a Wellcome Trust senior clinical fellow (WT088566, WT097820). F.S. has been funded by an ACT Clinical Research Training Fellowship and a joint Sparks-MRC Clinical Research Training Fellowship. C.-P.S. is funded by the Children's Liver Diseases Foundation.This is the final version of the article. It first appeared from Mary Ann Liebert Publishers via http://dx.doi.org/10.1089/scd.2014.051

A Novel Chemically Differentiated Mouse Embryonic Stem Cell-Based Model to Study Liver Stages of Plasmodium berghei.

Asymptomatic and obligatory liver stage (LS) infection of Plasmodium parasites presents an attractive target for antimalarial vaccine and drug development. Lack of robust cellular models to study LS infection has hindered the discovery and validation of host genes essential for intrahepatic parasite development. Here, we present a chemically differentiated mouse embryonic stem cell (ESC)-based LS model, which supports complete development of Plasmodium berghei exoerythrocytic forms (EEFs) and can be used to define new host-parasite interactions. Using our model, we established that host Pnpla2, coding for adipose triglyceride lipase, is dispensable for P. berghei EEF development. In addition, we also evaluated in-vitro-differentiated human hepatocyte-like cells (iHLCs) to study LS of P. berghei and found it to be a sub-optimal infection model. Overall, our results present a new mouse ESC-based P. berghei LS infection model that can be utilized to study the impact of host genetic variation on parasite development

A Novel Chemically Differentiated Mouse Embryonic Stem Cell-Based Model to Study Liver Stages of Plasmodium berghei.

Asymptomatic and obligatory liver stage (LS) infection of Plasmodium parasites presents an attractive target for antimalarial vaccine and drug development. Lack of robust cellular models to study LS infection has hindered the discovery and validation of host genes essential for intrahepatic parasite development. Here, we present a chemically differentiated mouse embryonic stem cell (ESC)-based LS model, which supports complete development of Plasmodium berghei exoerythrocytic forms (EEFs) and can be used to define new host-parasite interactions. Using our model, we established that host Pnpla2, coding for adipose triglyceride lipase, is dispensable for P. berghei EEF development. In addition, we also evaluated in-vitro-differentiated human hepatocyte-like cells (iHLCs) to study LS of P. berghei and found it to be a sub-optimal infection model. Overall, our results present a new mouse ESC-based P. berghei LS infection model that can be utilized to study the impact of host genetic variation on parasite development

Phenotypic and functional analyses show stem cell-derived hepatocyte-like cells better mimic fetal rather than adult hepatocytes

Background & Aims: Hepatocyte-like cells (HLCs), differentiated from pluripotent stem cells by the use of soluble factors, can model human liver function and toxicity. However, at present HLC maturity and whether any deficit represents a true fetal state or aberrant differentiation is unclear and compounded by comparison to potentially deteriorated adult hepatocytes. Therefore, we generated HLCs from multiple lineages, using two different protocols, for direct comparison with fresh fetal and adult hepatocytes. Methods: Protocols were developed for robust differentiation. Multiple transcript, protein and functional analyses compared HLCs to fresh human fetal and adult hepatocytes. Results: HLCs were comparable to those of other laboratories by multiple parameters. Transcriptional changes during differentiation mimicked human embryogenesis and showed more similarity to pericentral than periportal hepatocytes. Unbiased proteomics demonstrated greater proximity to liver than 30 other human organs or tissues. However, by comparison to fresh material, HLC maturity was proven by transcript, protein and function to be fetal-like and short of the adult phenotype. The expression of 81% phase 1 enzymes in HLCs was significantly upregulated and half were statistically not different from fetal hepatocytes. HLCs secreted albumin and metabolized testosterone (CYP3A) and dextrorphan (CYP2D6) like fetal hepatocytes. In seven bespoke tests, devised by principal components analysis to distinguish fetal from adult hepatocytes, HLCs from two different source laboratories consistently demonstrated fetal characteristics. Conclusions: HLCs from different sources are broadly comparable with unbiased proteomic evidence for faithful differentiation down the liver lineage. This current phenotype mimics human fetal rather than adult hepatocytes

Immunization with live, virulence-attenuated Listeria monocytogenes provides newborn mice with long-term protection against asthma

Asthma is a chronic respiratory disorder that leads to inflammation and narrowing of the airways. The global prevalence, morbidity, mortality and economic cost associated with asthma have been on the rise since the 1960’s and continue to increase dramatically by 50% every decade. Today, over 300 million people across all ages, genders and ethnic backgrounds suffer from asthma. In short, asthma has reached epidemic levels. Current treatment options either alleviate symptoms only temporarily, burden the asthmatic with life-long controller medication, or rely on environmental control measures. Children are disproportionally affected by asthma, indicating that asthma most often is initiated early in life. A vaccination strategy able to prevent or cure asthma early in life is therefore urgently needed. We have successfully developed a novel vaccine platform based on the live, attenuated, intracellular bacterium Listeria monocytogenes. We hypothesized that this vaccine platform would induce a sustained anti-allergic Th1 immune response after only one dose given to newborn mice, thus preventing asthma upon future challenge with the allergen. To test our hypothesis, neonatal mice immunized intraperitoneally with different Listeria monocytogenes vaccine strains were compared to negative and positive controls. We examined the protective effects of the following vaccines: the live vaccine strain Lm (trpS actA)/pSPOPShlyOVA, synthesizing ovalbumin proteins, 2) the same yet heatkilled vaccine strain HKLm (trpS actA)/pSPO-PShlyOVA and 3) the live vaccine strain Lm (trpS actA)/pSPO expressing no specific antigens. Subsequent sensitization and intranasal challenge with ovalbumin to induce asthma was followed by a detailed analysis of asthma severity. This analysis included the total number and types of cells in the bronchoalveolar lavage fluid, as well as histology of lung tissue identifying goblet cell metaplasia and cell infiltration of the airway epithelium. Serum antibody levels and cytokine profiles were also examined, as was airway resistance. We found that only neonatal mice immunized with the live Listeria monocytogenes strains were protected from asthma. This protection did not appear to be mediated by shifts in the Th1/Th2 responses and was found even in the absence of specific antigen expression by Listeria monocytogenes.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Proteomic comparison of various hepatic cell cultures for preclinical safety pharmacology

Experimental drugs need to be screened for safety within time constraints. Hepatotoxicity is one concerning contributor to the failure of investigational new drugs and a major rationale for postmarketing withdrawal decisions. Ethical considerations in preclinical research force the requirement for highly predictive in vitro assays using human tissue which retains functionality reflective of primary tissue. Here, the proteome of cells commonly used to assess preclinical hepatotoxicity was compared. Primary human hepatocytes (PHHs), hepatocyte-like cells (HLCs) differentiated from human pluripotent stem cells, HepG2 cell monolayers and HepG2 cell 3D spheroids were cultured and collected as whole cell lysates. Over 6000 proteins were identified and quantified in terms of relative abundance in replicate proteomic experiments using isobaric tagging methods. Comparison of these quantitative data provides biological insight into the feasibility of using HLCs, HepG2 monolayers, and HepG2 3D spheroids for hepatotoxicity testing. Collectively these data reveal how HLCs differentiated for 35 days and HepG2 cells proteomes differ from one another and that of PHHs. HepG2 cells possess a strong cancer cell signature and do not adequately express key metabolic proteins which mark the hepatic phenotype, this was not substantially altered by culturing as 3D spheroids. These data suggest that while no single hepatic model reflects the diverse array of outcomes required to mimic the in vivo liver functions, that HLCs are the most suitable investigational avenue for replacing PHHs in vitro.This research was supported in part by the National Research Foundation of South Africa for the grant (Grant No. 87880.) TH was supported by a UK Commonwealth Split-site PhD Scholarship (ZACS-2014-653) and a Commonwealth, European and International Cambridge Trust Scholarship (USN: 302989247; App No: 10326363). CPS was funded by Children Liver Disease foundation PhD studentship and LV by the ERC starting Grant Relieve IMD.http://toxsci.oxfordjournals.org2019-07-01hj2018Pharmacolog