Applying Pluripotent Stem Cells in Drug Discovery
genotype as the fibroblasts from which they were derived. The researchers were then successful in differentiating the cells into motor neurons of the type destroyed in ALS based on the expression of HB9 (a motor-neuron-specific transcription factor).
The ability to generate motor neurons affected by ALS may soon provide a potent tool for research into the biology underlying ALS and screening for new drugs. The study from Kevin Eggan’s laboratory demonstrated that the iPS cells generated from the ALS patient had a similar gene expression signature to that of hES cells. Moreover, the researchers demonstrated the potential of iPS technology in personalised, regenerative medicine thanks to the feasibility of producing large numbers of motor neurons with a patient’s exact genotype and immune match, which could be a source for transplantation. However, major hurdles will have to be overcome before this becomes a reality.
Just after these breakthroughs, the laboratory of George Daley reported that they had produced iPS cells from patients with 10 different genetic diseases, including Gaucher disease, type 1 diabetes, Parkinson’s disease and Huntington’s disease, providing a compelling illustration of the contribution iPS cells could make to cellular disease modelling.5
By contrast, producing disease-specific
cells from hES cells requires exact knowledge of the nature of the genetic disease and subsequent genetic modification of normal lines, or for the stem cells to be derived from embryos carrying an inherited disease. These are not productive methods, and few diseases have been modelled in this way.
Further reports of patient-specific cell lines generated by re-programming of adult cells to an induced pluripotent state and then differentiating them into a specific cell type have followed. Examples are dopaminergic neurons from Parkinson’s disease patients by Soldner et al.,6
atrophy (SMA) patient by Ebert et al.,7 familial dysautonomia patient by Lee et al.8
motor neurons from a spinal muscular peripheral neurons from a and disease-corrected
haematopoietic progenitors from Fanconi anaemia patients by Raya et al.9
The SMA study7
was the first to show that iPS cells can be used to model the specific pathology seen in a genetically inherited disease. The motor neurons generated from the SMA iPS cells showed characteristics associated with the disease, i.e. decrease in size and number during culturing, which were not observed in neurons derived from the child’s unaffected mother. In the study on familial dysautonomia,8
closely related to hES cells than is the case in iPS cells carrying transgenes.6
Thus, technologies are advancing at a rapid pace, and in the future it will be a challenge to standardise directed differentiation protocols in order to compare between-laboratory results.
In showing that it is possible to correct the genetic defect in iPS cells from Fanconi anaemia patients and then differentiate them into haematopoietic progenitor cells, Raya et al. provided proof-of-concept that iPS technology can be used for the generation of disease- corrected patient-specific cells of potential value for cell therapy.9
Issues in Applying Induced Pluripotent Stem Cells in Disease Modelling
Research to date has raised expectations that taking adult cells from a patient, re-programming them to iPS cells and then differentiating them to the cell type that is affected in a disease could provide models of unprecedented power in drug discovery. Furthermore, it has been shown that cells can be directly re-programmed to other cell types without going through the induced pluripotent state. Vierbuchen et al. demonstrated that mouse fibroblasts can be directly re-programmed to functional neurons without first deriving iPS cells.10 These induced neuronal cells express multiple neuron-specific proteins, generate action potentials and form functional synapses. Generation of neuronal cells, either via iPS cells or directly from other types of cell, could have important implications for studies of neural development and disease modelling, since it is impossible to obtain live samples of diseased tissue. In this example, having patient- specific neurons could provide a system for investigating molecular and cellular changes that are implicated in each disease. In Parkinson’s this would include, in cultured dopaminergic neurons, neuronal dysfunction, protein aggregation, mitochondrial defects, oxidative stress and defects in signal transduction.
The issue, of course, is looking at these processes along the progression of the disease. It is not yet clear how and if the Parkinson’s disease genotype from an adult cell transmits into a disease phenotype of a dopaminergic cell differentiated from an iPS cell. There are other nuts and bolts technical issues to be resolved. For a start, the re-programming process remains highly inefficient, and the reasons for this remain unclear. Research is in progress to replace viral or DNA vectors with small molecules or human proteins. In May 2009 Kim et al.11
the utility of iPS cells in disease modelling was very much expanded: three easily quantifiable parameters of the disease state were identified, comprising both molecular and cellular read- outs, which could then be exploited to monitor drug response. These studies demonstrated the huge potential of iPS technology to monitor and understand the pathophysiology of disease.
Meanwhile, the research in Parkinson’s disease by Soldner et al. represented a further advance in that the genes used to re-programme the adult cells were delivered by viral vectors that were subsequently excised from the resulting iPS cells. This is important because expression of the transgenes could alter the differentiation potential of the iPS cells or induce malignant transformation. Importantly, Soldner et al. showed that the iPS cells maintained their pluripotent characteristics after the re-programming factors were removed. These cells also had a global gene expression profile more
DRUG DISCOVERY
reported re-programming human fibroblasts with four recombinant proteins without the use of viral vectors, while Lin et al.12 described how they had re-programmed skin cells using three small molecules. Another issue to be addressed in creating robust models from patient-specific iPS cells is how to mimic disease processes that occur over many years. It may be that disease pathology will not manifest itself in vitro unless new techniques are developed for artificially ageing differentiated cell lines.
It is also clear that, at best, working with single cell types will only provide a partial picture of what is happening: diseased cells are likely to interact with other types of cell in making the symptoms of disease manifest. Realistic in vitro models may thus require more than one type of cell. For example, in Parkinson’s disease this implies not only the dopaminergic neurons but also the surrounding glia cells, including astrocytes and oligodendrocytes, as well as the immune cells that contribute to neuronal inflammation. It may turn out that cells from iPS cells are only truly useful in modelling inherited monogenetic disease.
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