Comparative transcriptomics of hepatic differentiation of human pluripotent stem cells and adult human liver tissue.

Hepatocytes derived from human pluripotent stem cells (hPSC-HEP) have the potential to replace presently used hepatocyte sources applied in liver disease treatment and models of drug discovery and development. Established hepatocyte differentiation protocols are effective and generate hepatocytes, which recapitulate some key features of their in vivo counterparts. However, generating mature hPSC-HEP remains a challenge. In this study, we applied transcriptomics to investigate the progress of in vitro hepatic differentiation of hPSCs at the developmental stages, definitive endoderm, hepatoblasts, early hPSC-HEP, and mature hPSC-HEP, to identify functional targets that enhance efficient hepatocyte differentiation. Using functional annotation, pathway and protein interaction network analyses, we observed the grouping of differentially expressed genes in specific clusters representing typical developmental stages of hepatic differentiation. In addition, we identified hub proteins and modules that were involved in the cell cycle process at early differentiation stages. We also identified hub proteins that differed in expression levels between hPSC-HEP and the liver tissue controls. Moreover, we identified a module of genes that were expressed at higher levels in the liver tissue samples than in the hPSC-HEP. Considering that hub proteins and modules generally are essential and have important roles in the protein-protein interactions, further investigation of these genes and their regulators may contribute to a better understanding of the differentiation process. This may suggest novel target pathways and molecules for improvement of hPSC-HEP functionality, having the potential to finally bring this technology to a wider use.

Preclinical imaging methods for assessing the safety and efficacy of regenerative medicine therapies.

Regenerative medicine therapies hold enormous potential for a variety of currently incurable conditions with high unmet clinical need. Most progress in this field to date has been achieved with cell-based regenerative medicine therapies, with over a thousand clinical trials performed up to 2015. However, lack of adequate safety and efficacy data is currently limiting wider uptake of these therapies. To facilitate clinical translation, non-invasive in vivo imaging technologies that enable careful evaluation and characterisation of the administered cells and their effects on host tissues are critically required to evaluate their safety and efficacy in relevant preclinical models. This article reviews the most common imaging technologies available and how they can be applied to regenerative medicine research. We cover details of how each technology works, which cell labels are most appropriate for different applications, and the value of multi-modal imaging approaches to gain a comprehensive understanding of the responses to cell therapy in vivo.

Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines.

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.

High Content Analysis of Human Pluripotent Stem Cell Derived Hepatocytes Reveals Drug Induced Steatosis and Phospholipidosis.

Hepatotoxicity is one of the most cited reasons for withdrawal of approved drugs from the market. The use of nonclinically relevant in vitro and in vivo testing systems contributes to the high attrition rates. Recent advances in differentiating human induced pluripotent stem cells (hiPSCs) into pure cultures of hepatocyte-like cells expressing functional drug metabolizing enzymes open up possibilities for novel, more relevant human cell based toxicity models. The present study aimed to investigate the use of hiPSC derived hepatocytes for conducting mechanistic toxicity testing by image based high content analysis (HCA). The hiPSC derived hepatocytes were exposed to drugs known to cause hepatotoxicity through steatosis and phospholipidosis, measuring several endpoints representing different mechanisms involved in drug induced hepatotoxicity. The hiPSC derived hepatocytes were benchmarked to the HepG2 cell line and generated robust HCA data with low imprecision between plates and batches. The different parameters measured were detected at subcytotoxic concentrations and the order of which the compounds were categorized (as severe, moderate, mild, or nontoxic) based on the degree of injury at isomolar concentration corresponded to previously published data. Taken together, the present study shows how hiPSC derived hepatocytes can be used as a platform for screening drug induced hepatotoxicity by HCA.

One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells.

Human hepatocytes display substantial functional inter-individual variation regarding drug metabolizing functions. In order to investigate if this diversity is mirrored in hepatocytes derived from different human pluripotent stem cell (hPSC) lines, we evaluated 25 hPSC lines originating from 24 different donors for hepatic differentiation and functionality. Homogenous hepatocyte cultures could be derived from all hPSC lines using one standardized differentiation procedure. To the best of our knowledge this is the first report of a standardized hepatic differentiation procedure that is generally applicable across a large panel of hPSC lines without any adaptations to individual lines. Importantly, with regard to functional aspects, such as Cytochrome P450 activities, we observed that hepatocytes derived from different hPSC lines displayed inter-individual variation characteristic for primary hepatocytes obtained from different donors, while these activities were highly reproducible between repeated experiments using the same line. Taken together, these data demonstrate the emerging possibility to compile panels of hPSC-derived hepatocytes of particular phenotypes/genotypes relevant for drug metabolism and toxicity studies. Moreover, these findings are of significance for applications within the regenerative medicine field, since our stringent differentiation procedure allows the derivation of homogenous hepatocyte cultures from multiple donors which is a prerequisite for the realization of future personalized stem cell based therapies.

MicroRNA-122: a novel hepatocyte-enriched in vitro marker of drug-induced cellular toxicity.

Emerging hepatic models for the study of drug-induced toxicity include pluripotent stem cell-derived hepatocyte-like cells (HLCs) and complex hepatocyte-non-parenchymal cellular coculture to mimic the complex multicellular interactions that recapitulate the niche environment in the human liver. However, a specific marker of hepatocyte perturbation, required to discriminate hepatocyte damage from non-specific cellular toxicity contributed by non-hepatocyte cell types or immature differentiated cells is currently lacking, as the cytotoxicity assays routinely used in in vitro toxicology research depend on intracellular molecules which are ubiquitously present in all eukaryotic cell types. In this study, we demonstrate that microRNA-122 (miR-122) detection in cell culture media can be used as a hepatocyte-enriched in vitro marker of drug-induced toxicity in homogeneous cultures of hepatic cells, and a cell-specific marker of toxicity of hepatic cells in heterogeneous cultures such as HLCs generated from various differentiation protocols and pluripotent stem cell lines, where conventional cytotoxicity assays using generic cellular markers may not be appropriate. We show that the sensitivity of the miR-122 cytotoxicity assay is similar to conventional assays that measure lactate dehydrogenase activity and intracellular adenosine triphosphate when applied in hepatic models with high levels of intracellular miR-122, and can be multiplexed with other assays. MiR-122 as a biomarker also has the potential to bridge results in in vitro experiments to in vivo animal models and human samples using the same assay, and to link findings from clinical studies in determining the relevance of in vitro models being developed for the study of drug-induced liver injury.

Long-term chronic toxicity testing using human pluripotent stem cell-derived hepatocytes.

Human pluripotent stem cells (hPSC) have the potential to become important tools for the establishment of new models for in vitro drug testing of, for example, toxicity and pharmacological effects. Late-stage attrition in the pharmaceutical industry is to a large extent caused by selection of drug candidates using nonpredictive preclinical models that are not clinically relevant. The current hepatic in vivo and in vitro models show clear limitations, especially for studies of chronic hepatotoxicity. For these reasons, we evaluated the potential of using hPSC-derived hepatocytes for long-term exposure to toxic drugs. The differentiated hepatocytes were incubated with hepatotoxic compounds for up to 14 days, using a repeated-dose approach. The hPSC-derived hepatocytes became more sensitive to the toxic compounds after extended exposures and, in addition to conventional cytotoxicity, evidence of phospholipidosis and steatosis was also observed in the cells. This is, to the best of our knowledge, the first report of a long-term toxicity study using hPSC-derived hepatocytes, and the observations support further development and validation of hPSC-based toxicity models for evaluating novel drugs, chemicals, and cosmetics.

Drug metabolizing enzyme and transporter protein profiles of hepatocytes derived from human embryonic and induced pluripotent stem cells.

Human embryonic and induced pluripotent stem cell-derived hepatocytes (hESC-Hep and hiPSC-Hep) have the potential to provide relevant human in vitro model systems for toxicity testing and drug discovery studies. In this study, the expression and function of important drug metabolizing cytochrome P450 (CYP) enzymes and transporter proteins in hESC-Hep and hiPSC-Hep were compared to cryopreserved human primary hepatocytes (hphep) and HepG2 cells. Overall, CYP activities in hESC-Hep and hiPSC-Hep were much lower than in hphep cultured for 4 h, but CYP1A and 3A activities were comparable to levels in hphep cultured for 48h (CYP1A: 35% and 26% of 48 h hphep, respectively; CYP3A: 80% and 440% of 48 h hphep, respectively). Importantly, in hESC-Hep and hiPSC-Hep, CYP activities were stable or increasing for at least one week in culture which was in contrast to the rapid loss of CYP activities in cultured hphep between 4 and 48 h after plating. With regard to transporters, in hESC-Hep and hiPSC-Hep, pronounced NTCP activity (17% and 29% of 4 h hphep, respectively) and moderate BSEP activity (6% and 8% of 4 h hphep, respectively) were observed. Analyses of mRNA expression and immunocytochemistry supported the observed CYP and transporter activities and showed expression of additional CYPs and transporters. In conclusion, the stable expression and function of CYPs and transporters in hESC-Hep and hiPSC-Hep for at least one week opens up the possibility to reproducibly perform long term and extensive studies, e.g. chronic toxicity testing, in a stem cell-derived hepatic system.

Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project.

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.

Growth-limiting role of endothelial cells in endoderm development.

Endoderm development is dependent on inductive signals from different structures in close vicinity, including the notochord, lateral plate mesoderm and endothelial cells. Recently, we demonstrated that a functional vascular system is necessary for proper pancreas development, and that sphingosine-1-phosphate (S1P) exhibits the traits of a blood vessel-derived molecule involved in early pancreas morphogenesis. To examine whether S1P(1)-signaling plays a more general role in endoderm development, S1P(1)-deficient mice were analyzed. S1P(1) ablation results in compromised growth of several foregut-derived organs, including the stomach, dorsal and ventral pancreas and liver. Within the developing pancreas the reduction in organ size was due to deficient proliferation of Pdx1(+) pancreatic progenitors, whereas endocrine cell differentiation was unaffected. Ablation of endothelial cells in vitro did not mimic the S1P(1) phenotype, instead, increased organ size and hyperbranching were observed. Consistent with a negative role for endothelial cells in endoderm organ expansion, excessive vasculature was discovered in S1P(1)-deficient embryos. Altogether, our results show that endothelial cell hyperplasia negatively influences organ development in several foregut-derived organs.

Interwoven four-compartment capillary membrane technology for three-dimensional perfusion with decentralized mass exchange to scale up embryonic stem cell culture.

We describe hollow fiber-based three-dimensional (3D) dynamic perfusion bioreactor technology for embryonic stem cells (ESC) which is scalable for laboratory and potentially clinical translation applications. We added 2 more compartments to the typical 2-compartment devices, namely an additional media capillary compartment for countercurrent 'arteriovenous' flow and an oxygenation capillary compartment. Each capillary membrane compartment can be perfused independently. Interweaving the 3 capillary systems to form repetitive units allows bioreactor scalability by multiplying the capillary units and provides decentralized media perfusion while enhancing mass exchange and reducing gradient distances from decimeters to more physiologic lengths of <1 mm. The exterior of the resulting membrane network, the cell compartment, is used as a physically active scaffold for cell aggregation; adjusting intercapillary distances enables control of the size of cell aggregates. To demonstrate the technology, mouse ESC (mESC) were cultured in 8- or 800-ml cell compartment bioreactors. We were able to confirm the hypothesis that this bioreactor enables mESC expansion qualitatively comparable to that obtained with Petri dishes, but on a larger scale. To test this, we compared the growth of 129/SVEV mESC in static two-dimensional Petri dishes with that in 3D perfusion bioreactors. We then tested the feasibility of scaling up the culture. In an 800-ml prototype, we cultured approximately 5 x 10(9) cells, replacing up to 800 conventional 100-mm Petri dishes. Teratoma formation studies in mice confirmed protein expression and gene expression results with regard to maintaining 'stemness' markers during cell expansion.

Dynamic 3D culture promotes spontaneous embryonic stem cell differentiation in vitro.

Spontaneous in vitro differentiation of mouse embryonic stem cells (mESC) is promoted by a dynamic, three-dimensional (3D), tissue-density perfusion technique with continuous medium perfusion and exchange in a novel four-compartment, interwoven capillary bioreactor. We compared ectodermal, endodermal, and mesodermal immunoreactive tissue structures formed by mESC at culture day 10 with mouse fetal tissue development at gestational day E9.5. The results show that the bioreactor cultures more closely resemble mouse fetal tissue development at gestational day E9.5 than control mESC cultured in Petri dishes.

Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells.

Human embryonic stem cells (hESC) offer a potential unlimited source for functional human hepatocytes, since they can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing several hepatic markers. Such cells could be used for, e.g. studies of drug metabolism and hepatotoxicity, which however would require a significant expression of drug metabolising enzymes. Thus, we have investigated the expression of cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs), drug transporters, transcription factors and other liver specific genes in hepatocyte-like cells derived from hESC using a simple direct differentiation protocol. The mRNA and protein expression of several important CYPs were determined using low density arrays, real time PCR and Western blotting. Significant CYP expression on the mRNA level was detected in hepatocyte-like cells derived from one out of two different hESC lines tested, which was much higher than in undifferentiated hESC and generally higher than in HepG2 cells. CYP1A2, CYP3A4/7 and low levels of CYP1A1 and CYP2C8/9/19 protein were detected in both lines. The mRNAs for a variety of CYPs and liver specific factors were shown to be inducible in both cell lines, and this was reflected in induced levels of CYP1A2 and CYP3A4/7 protein. This first report on expression of all major CYPs in hepatocyte-like cells derived from hESC represents an important step towards functional hepatocytes, but efforts to further differentiate the cells using optimized protocols are needed before they exhibit similar levels of drug metabolizing enzymes as primary human hepatocytes and liver.

Glutathione transferases in hepatocyte-like cells derived from human embryonic stem cells.

Human embryonic stem cells (hESCs) offer a potential unlimited source for functional human hepatocytes, since hESCs can differentiate into hepatocyte-like cells displaying a characteristic hepatic morphology and expressing several hepatic markers. These hepatocyte-like cells could be used in various human in vitro hepatocyte assays, e.g. as a test system for studying drug metabolism and drug-induced hepatotoxicity. Since the toxic effect of a compound is commonly dependent on biotransformation into metabolites, the presence of drug metabolising enzymes in potential test systems must be evaluated. We have investigated the presence of glutathione transferases (GSTs) in hepatocyte-like cells by immunocytochemistry and Western blotting. Results show that these cells have high levels of GSTA1-1, whereas GSTP1-1 is not present in most cases. GSTM1-1 is detected by immunocytochemistry but not by Western blotting. In addition, GST activity is detected in hepatocyte-like cells at levels comparable to human hepatocytes. These results indicate that the hepatocyte-like cells have characteristics that closely resemble those of human adult hepatocytes.

Signals from the embryonic mouse pancreas induce differentiation of human embryonic stem cells into insulin-producing beta-cell-like cells.

The recent success in restoring normoglycemia in type 1 diabetes by islet cell transplantation indicates that cell replacement therapy of this severe disease is achievable. However, the severe lack of donor islets has increased the demand for alternative sources of beta-cells, such as adult and embryonic stem cells. Here, we investigate the potential of human embryonic stem cells (hESCs) to differentiate into beta-cells. Spontaneous differentiation of hESCs under two-dimensional growth conditions resulted in differentiation of Pdx1(+)/Foxa2(+) pancreatic progenitors and Pdx1(+)/Isl1(+) endocrine progenitors but no insulin-producing cells. However, cotransplantation of differentiated hESCs with the dorsal pancreas, but not with the liver or telencephalon, from mouse embryos resulted in differentiation of beta-cell-like cell clusters. Comparative analysis of the basic characteristics of hESC-derived insulin(+) cell clusters with human adult islets demonstrated that the insulin(+) cells share important features with normal beta-cells, such as synthesis (proinsulin) and processing (C-peptide) of insulin and nuclear localization of key beta-cell transcription factors, including Foxa2, Pdx1, and Isl1.

Vascular function and sphingosine-1-phosphate regulate development of the dorsal pancreatic mesenchyme.

Early growth and differentiation of the pancreatic endoderm is regulated by soluble factors from the pancreatic mesenchyme. Previously, we demonstrated that N-cadherin-deficient mice lack a dorsal pancreas, due to a critical role of N-cadherin in dorsal pancreatic mesenchymal cell survival. Here, we show that restoring cardiac and circulatory function in N-cadherin null mice by cardiac-specific expression of N-cadherin, rescues formation of the dorsal pancreas, indicating that the phenotype is secondary to defects related to cardiac/vascular function. Based on this observation, we demonstrate that soluble factors present in plasma, such as sphingosine-1-phosphate, rescue formation of the dorsal pancreas in N-cadherin-deficient mice. We also show that sphingosine-1-phosphate indirectly promotes budding of the pancreatic endoderm by stimulating pancreatic mesenchymal cell proliferation. Finally, we identify sphingosine-1-phosphate receptors within the mesenchyme and show that pertussis toxin blocks the sphingosine-1-phosphate-induced actions, suggesting the involvement of G-protein-coupled sphingosine-1-phosphate receptors. Thus, we propose a new model where blood vessel-derived sphingosine-1-phosphate stimulates growth and budding of the dorsal pancreatic endoderm by induction of mesenchymal cell proliferation.

Expression of dominant negative cadherin in the adult mouse brain modifies rearing behavior.

The cadherin superfamily of cell-cell adhesion molecules (CAM) are crucial regulators of morphogenesis and axonal guidance during development of the nervous system and have been suggested to play important roles in neural plasticity of the brain. To study the latter, we created a mouse model that expressed a dominant negative classical cadherin in the brain of adult mice. The mice were tested for spontaneous motor activity and exploratory behavior in the open field, anxiety in the plus-maze, and spatial learning and memory in the water-T maze. Mice expressing the dominant negative cadherin displayed reduced rearing behavior, but no change in motor activity, in the open field, indicating deficits in exploratory behavior. In the water maze, animals expressing the mutant cadherin showed normal escape latencies and were indistinguishable from control littermates. Similarly, LTP in hippocampal slices of cadherin mutant and control mice were indistinguishable. These findings demonstrate intact spatial learning in mice expressing a dominant negative cadherin but altered rearing behavior, suggesting the involvement of classical cadherins in mechanisms mediating rearing behavior.