I. Nuclear reprogramming and changing cell fate
It is now possible to modulate cell fate, to produce the desired somatic, multipotent or pluripotent cell type. We are now developing novels methods to both improve the current state-of-the-art, which rely heavily on integrative approaches that perturb the genome. In turn we will utilise these tools to dissect the molecular mechanisms of nuclear reprogramming. In particular, we are establishing methods to direct hESCs and hiPSCs to non-parenchymal hepatic cell fates.
II. Understanding endodermal differentiation
A major challenge faced by researchers is to produce hepatocyte like cells (HLCs) from pluripotent stem cells (PSCs) that faithfully recapitulate primary human hepatocytes in terms of their metabolic potential, particularly with respect to cytochrome p450 activity. We have developed a procedure to differentiate hPSC lines to functional hepatocytes which exhibit basal metabolism, production of serum proteins, key markers such as albumin and recapitulation of disease in a dish (Sullivan et al., 2009), but with the aforementioned limitations. In order to elucidate these functional differences we are charactering the products of the differentiation process from hPSC lines, at the transcriptional, microRNA and global chromatin level. An important component is to ensure highly enriched cell populations, which is achieved by utilising stage specific markers in conjunction with FACs sorting or by genome engineering (CRISPR/CAS) to produce lineage specific reporters representative of each time point of the differentiation, thus reducing the effect of noise. We will be compare our findings to foetal and primary human hepatocytes and this should provide insight into the molecular mechanisms required to reach an adult-like phenotype. HLCs, like other hPSC progeny have a foetal phenotype, the above study will potentially provide the molecular nature of this state and allow us to manipulate HLCs at the molecular level to coax a more mature phenotype.
III. The use of small molecules for the derivation of hepatic cell types from human pluripotent stem cells.
Human induced pluripotent stem cells (iPSCs) have significant potential to revolutionise medicine by offering the potential to model a wide range of diseases and genotypes in predictive toxicology; reducing the bottleneck leading to potential novel targets and cost effective clinical deployment. However, potential exploitation of hiPSCs and indeed hESCs in these fields are impeded by the inability to isolate large numbers of somatic/progenitor cells rapidly, reproducibly and cost-effectively. Current techniques utilise exogenous growth factors to drive differentiation, which represents a significant cost for even small-scale laboratory use. In addition, growth factors are rapidly degraded and therefore unsuitable for derivation of hepatocytes and other cell types on a large scale as required by Pharma. Hepatocytes are in high demand for drug testing, therefore the ability to design, test and optimise small molecules mimicking the desired biological activity of the relevant growth factors will allow cost-effective and quality assured production of hPSC derived somatic cells. Additionally, new compounds will allow the dissection of mode of action of small molecules in the differentiation process.
IV. 3D culture systems to develop physiologically relevent tissue models
Current cell culture models used to interrogate disease and toxicology rely on conventional 2D static culture systems. In many cases immortalized or tumour derived cell lines are used, these have lost many of the characteristics of the cell type they are meant to model due to extensive time in culture, which leads to particular homogeneous populations of cells being selected. A more relevant model system is based on primary cells, which have not been exposed to the selective pressures of conventional cell systems described above. But these systems have their innate inadequacies, for example freshly isolated 2D primary hepatocyte monocultures on plastic or collagen rapidly lose polarity and differentiated function. This is a consequence of lose their surrounding 3D micro-environment, which comprises the extracellular matrix (ECM) and interactions with other crucial non-parenchymal cell types. This phenomenon is not unique to hepatocytes, it has been observed in a number of other primary culture systems including cardiac and kidney. Therefore using advanced tissue engineering (TE) enabling technologies we will arrange stem cell derived progeny in 3-dimensional space to closely resemble, in physiology and composition, for example a functional liver sinusoid. The creation of this ‘3D liver organoid’ will be developed as to be amenable for high throughput studies of liver function. The physiologically relevant, 3D human organotypic models; offer a considerable advance in medical research and an important alternative to animal studies. Additionally by developing in vitro relevant 3D tissue models that recapitulate their native counterparts, these can be translated to the pharmaceutical arena with 3D organotypic liver models to interrogate diseases such as metabolic disorders and infectious agents such as hepatitis. These platforms could be utilised to investigate toxicology and help reduce drug attrition rates with more physiologically relevant systems.
V. Disease modelling
Mouse models of human diseases are invaluable tools but do not always faithfully mimic the human situation. Where the mouse and human physiology are different or indeed the genetic basis of the disease is unknown, human disease-speciﬁc iPSCs lines can be generated and differentiated into the tissues afflicted with the phenotype. Such model systems will undoubtedly provide unique insights into the disease pathophysiology. We are currently involved in the following disease models:
Mitochondrial DNA Polymerase gamma mutations, including Alpers Syndrome, in collaboration with Prof. Laurence Bindoff (University of Bergen).
Inborn errors of metabolism, including Niemann Pick disease, Alpha-1-antitrypsin deficiency (conformational proteinopathy), in collaboration with Dr. Yngve Bliksrud, Dr. Runar Almaas, Dr. Terje Rootwelt (Rikshospital, Oslo).
Neurodegenerative diseases, Schizophrenia, Bipolar disorder and Ataxias in collaboration with Prof. Ole Andreassan, Dr. Srjdan Djorvic (Intitute of Clinical Medicine, UiO) and Prof. Joel Glover.
VI. Conservation of critically endangered species
We are generating a bank of iPSCs from critically endangered species. We have successfully generated iPSCs from a number of critically endangered species via integration free methodologies producing “clean” iPSCs. This will provide an extremely powerful resource that will potentially allow us to generate gametes for assisted reproduction of these species.