News From The Cutting Edge Table 1: Advantages and Disadvantages of Human Cellular Models of Parkinson’s Disease2 Cell Type


Immortalised or cancer cell lines (e.g. SH-SY5Y, EBV-transformed lymphocytes, LUHMES)


Primary cell lines (e.g. fibroblasts, cybrid cells) Stem cells and induced pluripotent stem cells Advantages


• Low cost for maintenance • Homogenous cell population


• Fully differentiated cell types


• Close approximation of native function, but not necessarily the tissue type affected


• High quality • Available source of all cell types • Close approximation of native function Disadvantages • Lack important aspects of native function


(e.g. metabolic function and electrical properties)


• Not easily accessible or available for all cell types • Questionable reproducibility


• Maintenance is expensive


• Time-consuming to obtain fully differentiated cell types


• Require more effort to achieve purified population


EBV = Epstein-Barr virus; LUHMES = Lund human mesencephalic. Figure 1: Modelling ‘Parkinson’s Disease in a Dish’


Skin cells


Skin biopsy from a Parkinson’s patient, isolation and growth of skin cells


iPSC colony Nuclear reprogramming


conditions for two to six weeks until stem cell-like colonies appear. These are then manually picked and characterised for markers of pluripotency and differentiation potential.21


Neuronal rossettes Neural stem cells


Directed neuronal differentation


Mature neurons


Transcription factors and genetic delivery methods that are used for the induction of iPSCs may vary, but certain key regulators such as Oct4 cannot be substituted. Initial nuclear reprogramming strategies utilised viral vectors with the caveat that they either do not silence completely or reactivate at a later state, thus hampering the potential to differentiate in specific tissue types. The addition of transcription factors also has the theoretical risk of producing cancer cells. To address this, several factor-free strategies have been proposed for the purpose of nuclear reprogramming, including excision of the reprogramming vector, use of proteins or small molecules (non-DNA strategy), or vectors that do not integrate into the host genome (non-integration strategy).22,23


Study of disease mechanisms and in vitro ‘clinical trials’ to find drugs that reverse symptoms of Parkinson’s disease


iPSC = induced pluripotent stem cell.


Embryonic stem cells from non-human primates derived through cloning was first accomplished a decade later in 2007.7 In contrast, cell fusion, e.g. heterokaryons of mouse embryonic stem cells and human fibroblasts, is another notable technique for reprogramming that is specifically useful for understanding the regulatory programmes that are involved in this process.8–10


of a somatic cell, which can form an entire organism. This was successfully demonstrated by cloning of the first mammal, Dolly the sheep, in 1997.6


Mechanism of Nuclear Reprogramming iPSCs are very similar in their expression pattern to embryonic stem cells. They express pluripotency genes at high levels and lineage- specific genes are suppressed. Over multiple passages, the expression signature of iPSCs become even more similar to embryonic stem cells.24


epigenetic landscape25


Nuclear reprogramming requires a dramatic alteration of the and only a few mouse iPSC clones are


completely reprogrammed and suitable for producing a completely iPSC-derived offspring, apparently due to a single imprinted gene cluster Dlk1-Dio3.26


One of the key epigenetic regulators is the


Transcription factor-based nuclear reprogramming relies on the introduction of exogenous factors into somatic cells that reprogramme or ‘rejuvenate’ via modification of their epigenetic signature to a state almost indistinguishable from embryonic stem cells. Nuclear reprogramming has been shown to be successful for a variety of adult somatic tissues from all three germ cells, including fibroblasts after more than 20 passages,11


gingival cells,12 mesentery-derived cells,15 keratinocytes,13 peripheral blood cells16–18 hepatocytes,14 gut and amniocytes.19,20


Using this technique, a model for ‘PD in a dish’ can be developed (see Figure 1). In simple terms, skin cells from a patient with PD are isolated and expanded in culture for eight to 10 weeks. Depending on the protocol, a combination of specific transcription factors (for example Oct4, Sox2, Klf4 and c-Myc) is introduced into these patient-specific cells, which are then cultured under stem cell


28


Differentiation into Functional Dopaminergic Neurons


The derived patient-specific iPSCs lay the foundation for differentiation into the tissue-type of interest, i.e. mid-brain dopaminergic neurons that are specifically vulnerable and subject to neurodegeneration in PD. A large body of literature is accumulating on protocols that differentiate embryonic stem cells, or more recently iPSCs, into dopaminergic neurons. The driving force behind this


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activation-induced cytidine deaminase (AID) which acts as a DNA demethylase and ‘aids’ nuclear reprogramming, as shown in very elegant cell fusion experiments of human fibroblasts and mouse embryonic stem cells.8


Another epigenetic mark that is crucial for gene


activation/silencing is histone modification. Reprogrammed iPSCs re-acquire bivalent marks of H3K4 and H3K27 trimethylation, as in embryonic stem cells, thus reactivating expression of pluripotent genes.27,28


The completion of nuclear reprogramming is complex, but forward-steps are being made to unravel this astounding process.


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