Chris Denning's lab works on human pluripotent stem cells (hPSC), including human embryonic stem cells (hESC; derived from the inner cell mass of the pre-implantation embryo) and human induced pluripotent stem cells (hiPSC; derived by reprogramming somatic cells with genetic factors). We have produced 2 hESC lines (NOTT1 and NOTT2) that are deposited and distributed by the UK Stem Cell Bank. We have a total of ~20 hESC lines in-house and have produced more than 30 hiPSC lines in-house. We are using these lines to improve hPSC technology, including in automated scale up and in producing synthetic substrates. A central theme is to differentiate hPSCs into cardiomyocytes for drug screening and disease modelling, coupled with genetic engineering using the Cas9/CRISPR system
The lab is involved with standardisation of culture methods for hPSCs, as well as with genetic modification. Automated scale up using robotic platforms is an area of continued focus. We have demonstrated that our culture strategies are compatible with the CompacT SelecT, a robotic platform that can culture up to 90 x T175 flasks. More recently, we have purchased a completely bespoke Tecan Robotics platform that can culture 100 plates in a variety of formats from single well through to 384-well.
Another aspect of the work is to identify novel synthetic substrates for hPSC culture. Recent work showed that modification of the surface chemistry of tissue culture polystyrene by plasma etching turned a substrate not permissive for hPSC culture into a permissive one. This work is being extended to identify improved substrates. In this vein, we are producing libraries of 10,000 polymers that can be screening for suitability in hPSC culture using the Tecan robotic platform. We are also testing these substrates for their ability to direct differentiation of hPSCs to cardiomyocytes, hepatocytes and osteoblasts.
Our work on cardiomyocytes is directed towards improving differentiation efficiencies as 3D aggregates (embryoid bodies) and as 2D monolayers. This iterative process has now produced strategies where 70%+ of hPSCs are converted into cardiomyocytes. This is providing novel platforms for drug screening and disease modelling, including via production of hiPSC lines that carry mutations associated with structural, survivial and signalling disorders of the heart, such as Duchenne muscular dystrophy, myotonic dystrophy, Long QT Syndrome, CPVT, hypertrophic cardiomyopathy, beta-adrenoceptor signalling.
Expanded work areas:
Human Embryonic Stem Cells
Nottingham is one of 9 centres in the UK that have a license from the Human Fertilisation and Embryology Authority that allows them to derive stem cell lines from human embryos. This project represents a major collaboration with the University's fertility clinic, NURTURE. In this programme embryos donated by patients that are surplus to their treatment have already allowed the derivation of two new hESC lines that have been deposited into the UK Stem Cell bank to maximise their use. Researchers wishing to use NOTT1 and NOTT2 should contact the UK Stem Cell Bank.
Human Induced Pluripotent Stem Cells
Nottingham has a highly active programme in producing human induced pluripotent stem cells (hiPSCs) and we have over 30 lines derived from healthy individuals of those carrying genetic disorders of the heart (Dick et al., 2011a; Dick et al., 2011b; Matsa et al., 2011). The process we use starts by signing up patients by informed consent. This allows them to provide us with samples from skin, teeth, gums, hair or blood. From these samples, we isolate cells and introduce 4 genetic factors, which start conversion to hiPSCs. Once established, hiPSCs can be differentiated into beating heart cells (cardiomyocytes). These cells contain the same (damaged) genetic information as the patient and so provide an opportunity to model the disease and develop better therapies. We have produced models of several genetic diseases, including Duchene muscular dystrophy and Sudden Cardiac Death (see also Drug screening & disease modelling below).
Standardising Pluripotent Stem Cell Culture
Although in excess of 1800 human pluripotent stem cell lines (800 hESC lines & 1000 hiPSC lines) have been derived worldwide, there is considerable diversity in the methods used for culture the starting material (embryos or somatic cells), derivation method and passaging method. Diversity also extends to culture substrate and media formulation. Our earlier work standardised culture conditions such that at least 14 hPSC lines derived by different institutions could be maintained in the same feeder-free culture conditions using trypsin-passaging. These conditions retained karyotypic stability, as well as allowing standardised differentiation and genetic modification (Denning et al., 2006; Burridge et al., 2007; Anderson et al., 2007; Braam et al., 2008). The work also paved the way for development of new synthetic polymer-based growth substrates (Mahlstedt et al., 2010; see polymer / surface engineering below) and automated scale-up of hPSC culture (Thomas et al., 2009; also see scale-up below).
Polymer/ Surface Engineering
Current hPSC culture strategies typically rely on substrates that are undefined and / or show a high level of batch to batch variation. For example, human or mouse feeder cells are frequently used to provide a growth matrix. Alternatively, culture vessels are coated with products such as Matrigel, a solubulized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in laminin, collagen IV, heparan sulfate proteoglycans and entactin. Laminin, collagen and fibronectin have also been used. We first demonstrated that the surface chemistry tissue culture polystyrene (TCPS), which does not support hPSC culture, can be modified by radio frequency plasma etching, a process that produces highly reactive species similar to those found in ozone. This process produces plasma etched (PE) TCPS, which does allow culture of hPSCs (Mahlstedt et al., 2010) but in combination with a culture medium, which is poorly defined (i.e. conditioned medium). Current projects are now developing an extensive library of 10,000 polymers that will be used in robotic screens to identify the ones that support hPSC maintenance in defined medium. A spin off from this technology is the use of these polymers in the directed differentiation and maturation of hPSCs into cardiomyocytes, hepatocytes and osteblasts.
Scalability of hESCs represents a major barrier to realising commercial applications. In collaboration with Prof. David Williams and Dr. Rob Thomas (University of Loughborough), we have shown our hPSC culture protocols translate to the Compact SelecT, a fully automated system capable of handling up to 90 x T175 flasks. More recently, we have purchased a bespoked Tecan robotics platform that can handle 100 plates, ranging from single well to 384 well plates. We are using this to scale up hPSC culture and also automate hPSC differentiation to cardiomyocytes, hepatocytes and osteoblasts.
The ability to produce cardiomyocytes from hPSCs offers new opportunities in drug / toxicological screening, disease modelling and cell replacement therapies. Although hPSC-cardiomyocytes can be produced, the resulting cultures are highly heterogeneous with only the minority of the cells derived being cardiomyocytes. The two ways to improve this situation are to improve the differentiation efficiency and to enrich the cardiomyocytes that are present. Over the last 8 years, we have explored both strategies. We developed the technique of forced aggregation to form spontaneously differentiating embryoid bodies and showed that inclusion of the growth factors, FGF2 and activin A, greatly enhanced differentiation efficiencies (Burridge et al., 2007). In more recent work, we have gone on to show that further modification of the process can produce populations of embryoid bodies where 100% of the aggregates contain beating cardiomyocytes. An alternative route of differentiation is as monolayers rather than 3D embryoid bodies. We have shown that delivery of genetic factors (GATA4, TBX5, NKX2.5 and BAF60C) can induce high efficiency cardiomyocyte differentiation (Dixon et al., 2011), as can the use of the growth factors, BMP4 and activin A. To address enrichment, we engineered hPSCs to express drug resistance genes (puromycin N-acetyltransferase) specifically in differentiating cardiomyocytes. Treatment of the differentiated transgenic hPSCs with puromycin led to enrichment of cardiomyocytes to ~92% purity. Improvements in differentiation efficiency and purity have now allowed hPSC-cardiomyocytes to be used in drug screening and safety assessment, as well as disease modelling. For examples of the work we are doing in both areas see Drug screening and disease modelling below.
Drug development is costly; from bench to bedside for a single drug costs US$1.5 billion. Drug development within a pharmaceutical company starts with a million compound library screen (Stage 1) that identifies thousands of potential 'hits', which require efficacy and safety testing (Stage 2). A few candidates make it to preclinical and clinical testing (Stage 3), which associates with ~70% of development cost.
A major focus of Stage 2 is evaluating whether drugs harm the heart (cardiotoxicity). The problem is that there is no consistent supply of human cardiomyocytes that can be used. This means that Pharma relies on highly suboptimal models. These include the use of tumour cell lines that over express a single ion channel, which is far removed from functioning heart cells, where over 30 channels are expressed. Alternatively, primary cardiomyocytes from animals are used but these cells have different properties when compared to their human counterparts. Pharma needs better test systems. They must reduce the number of drugs unnecessarily entering Stage 3 and hence reduce the market withdrawal rate arising from unexpected cardiotoxicity/death in patients, which underlies the recent retraction of 8 non-cardiovascular drugs at a cost of US$12 billion.
Our recent work was in collaboration with the Stem Cells for Safer Medicine consortium (government plus GSK, AZ, Roche). This showed that drugs known to adversely affect the electrophysiology of the heart had the same affect in hPSC-derived cardiomyocytes (Dick et al., 2010). This work is continuing such that testing can be higher throughput and more informative.
Duchenne Muscular Dystrophy (DMD)
DMD is a fatal X-linked genetic disorder that affects ~1:3500 newborn males. Patients are typically wheelchair-bound by their early teens and succumb to cardiac or respiratory failure in their twenties. While the genetic lesion that causes the condition has been mapped to the DMD gene that encodes the protein dystrophin, the downstream molecular consequences are poorly defined. This is due both to suboptimal animal models (the mouse DMD gene knockout does not faithfully recapitulate the human condition) and to the inability to obtain human heart biopsies during progression of the disorder. We have produced six hiPSC lines from DMD patients (Dick et al., 2011). By deriving cardiomyocytes from these lines, we are evaluating whether the function of the cells can be restored by drug, RNA or gene therapy.
Sudden Cardiac Death
If mutations in the genome cause ion channels in the heart to function inappropriately, the rhythmic electrical waves that flow through the heart are disturbed. This means that the heart beat also becomes disturbed and an arrhythmia can occur. This can be fatal, hence the name Sudden Cardiac Death. A feature of some of these diseases is that the duration of the heart beat increase, which is known as Long QT Syndrome (LQTS). We have produce hiPSCs from patients with LQTS and can show that the electrical disturbances seen in the heart of these patients also occurs in the LQTS hiPSC-cardiomyocytes in the lab (Matsa et al., 2011). We have tested different drug treatments and, in each case, what occurs in the patient is mirrored in the LQTS hiPSC-cardiomyocytes. This is providing a new platform with which to evaluate new drugs and potentially develop new treatments for LQTS patients.
For development of many biotechnological applications, it is important to secure a patent to protect the intellectual property rights (IPR) of the invention. The United States patent office has granted many hESC-related patents, including the Wisconsin Alumni Research Foundation (WARF) patent, which has broad claims relating to many aspects of hESC and has restricted biotechnological commercialisation in many countries. In contrast, the European patent office has either stalled or denied all patents relating to hESC technologies on the grounds of Rule 23d(c) of the European Patent Convention, which prohibits the patenting of the "human embryo" on moral grounds. This has led to the filing via the patent offices of individual countries (i.e. the 'National Route') or to the development of hESC isolation technologies that may now require destruction of human embryos. These topics have been considered in detail by a European Union funded project, 'EU Stem Cell Patents', which the Commission is taking under advice. Details can be found in Porter et al., (2006).