Stabilisation of hepatocyte phenotype using synthetic materials

Primary human hepatocytes are a scare resource with limited lifespan and variable function which diminishes with time in culture. As a consequence, their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESC) could provide a stable source of human tissue due to their self-renewal properties and their ability to give rise to all the cell types of the human body. Therefore, hESC have the potential to provide an unlimited supply of hepatocytes. To date, the use of hESCs-derived somatic cells is limited due to the undefined, variable and xeno-containing microenvironment that influences the cell performance and life span, limiting scale-up and downstream application. Therefore, the development of highly defined cell based systems is required if the true potential of stem cell derived hepatocytes is to be realised. In order to replace the use of animal derived culture substrates to differentiate and maintain hESCs-derived hepatocytes, an interdisciplinary approach was employed to define synthetic materials, which maintain hepatocyte-like cell phenotype in culture. A simple polyurethane, PU134, was identified which improved hepatocyte performance and stability when compared to biological matrices. Moreover, the synthetic polymer was amenable to scale up and demonstrated batch-to-batch consistency. I subsequently used the synthetic polymer surface to probe the underlying biology, identifying key modulators of hepatocyte-like cell phenotype. This resulted in the identification of a novel genetic signature, MMP13, CTNND2 and THBS2, which was associated with stable hepatocyte performance. Importantly, those findings could be translated to two hESC lines derived at GMP. In conclusion, hepatocyte differentiation of pluripotent stem cells requires a defined microenvironment. The novel gene signature identified in this study represents an example of how to deliver stable hESCs-derived hepatocytes

Robust generation of hepatocyte-like cells from human embryonic stem cell populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies

Maintaining hepatic stem cell gene expression on biological and synthetic substrata

The liver is a highly resilient organ that possesses enormous regenerative capacity. This is mediated mainly through the most abundant cell type found in the liver, the hepatocyte. When the regenerative capacity of the hepatocyte is compromised, during chronic or acute liver injury, hepatic progenitor cells (HPCs) are activated to replace the damaged tissue. The HPC resides in a laminin-rich environment; as HPCs differentiate toward a hepatic or biliary fate, the extracellular matrix (ECM) composition changes, influencing cell behavior. To assess the impact that the biological ECM and the synthetic ECM have on the maintenance of hepatic stem cell gene expression, a murine hepatic stem cell line was employed. We demonstrate that hepatic stem cell gene expression could be maintained using a biological or synthetic substratum, but not on plastic alone

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines promising advances in cell-based modelling and therapies

Reducing hepatocyte injury and necrosis in response to paracetamol using non-coding RNAs

The liver performs multiple functions within the human body. It is composed of numerous cell types, which play important roles in organ physiology. Our study centers on the major metabolic cell type of the liver, the hepatocyte, and its susceptibility to damage during drug overdose. In these studies, hepatocytes were generated from a renewable and genetically defined resource. In vitro-derived hepatocytes were extensively profiled and exposed to varying levels of paracetamol and plasma isolated from liver-failure patients, with a view to identifying noncoding microRNAs that could reduce drug- or serum-induced hepatotoxicity. We identified a novel anti-microRNA, which reduced paracetamol-induced hepatotoxicity and glutathione depletion. Additionally, we identified a prosurvival role for anti-microRNA-324 following exposure to plasma collected from liver failure patients. We believe that these studies represent an important advance for the field, demonstrating the power of stem cell-derived systems to model human biology “in a dish” and identify novel noncoding microRNAs, which could be translated to the clinic in the future. SIGNIFICANCE: The liver performs vital functions within the human body and is composed of numerous cell types. The major metabolic cell type of the liver, the hepatocyte, is susceptible to damage during drug overdose. In these studies, hepatocytes were generated from a renewable resource and exposed to varying levels of paracetamol, with a view to identifying interventions that could reduce or attenuate drug-induced liver toxicity. A novel noncoding RNA that reduced paracetamol-induced hepatocyte toxicity was identified. These findings may represent an important advance for the field

Pluripotent Stem Cell-Derived Hepatocytes Inhibit T Cell Proliferation In Vitro through Tryptophan Starvation.

Regenerative medicine aims to replace damaged tissues by stimulating endogenous tissue repair or by transplanting autologous or allogeneic cells. Due to their capacity to produce unlimited numbers of cells of a given cell type, pluripotent stem cells, whether of embryonic origin or induced via the reprogramming of somatic cells, are of considerable therapeutic interest in the regenerative medicine field. However, regardless of the cell type, host immune responses present a barrier to success. The aim of this study was to investigate in vitro the immunological properties of human pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs). These cells expressed MHC class I molecules while they lacked MHC class II and co-stimulatory molecules, such as CD80 and CD86. Following stimulation with IFN-γ, HLCs upregulated CD40, PD-L1 and MHC class I molecules. When co-cultured with allogeneic T cells, HLCs did not induce T cell proliferation; furthermore, when T cells were stimulated via αCD3/CD28 beads, HLCs inhibited their proliferation via IDO1 and tryptophan deprivation. These results demonstrate that PSC-derived HLCs possess immunoregulatory functions, at least in vitro