Pathology in patients with familial Alzheimer’s disease patients and respective pluripotent stem cell (PSC) studies.
Neurodegenerative diseases are being modelled in-vitro using human patient-specific, induced pluripotent stem cells and transgenic embryonic stem cells to determine more about disease mechanisms, as well as to discover new treatments for patients. Current research in modelling Alzheimer’s disease, frontotemporal dementia and Parkinson’s disease using pluripotent stem cells is described, along with the advent of gene-editing, which has been the complimentary tool for the field. Current methods used to model these diseases are predominantly dependent on 2D cell culture methods. Outcomes reveal that only some of the phenotype can be observed in-vitro, but these phenotypes, when compared to the patient, correlate extremely well. Many studies have found novel molecular mechanisms involved in the disease and therefore elucidate new potential targets for reversing the phenotype. Future research that includes studying more complex 3D cell cultures, as well as accelerating aging of the neurons, may help to yield stronger phenotypes in the cultured cells. Thus, the use and application of pluripotent stem cells for modelling disease have already shown to be a powerful approach for discovering more about these diseases, but will lead to even more findings in the future as gene and cell culture technology continues to develop.
- Disease modelling
- Alzheimer’s disease
- frontotemporal dementia
- Parkinson’s disease
- pluripotent stem cells
The ability for researchers to model diseases in a dish has accelerated during the past decade, thanks to the discovery of a new stem cell type, the induced pluripotent stem cell (iPSC). This is an artificially created cell that recapitulates all the features of embryonic stem cells (ESCs) isolated from the early pre-implantation embryo. The production of this cell type in 2006 was a remarkable finding which led its founder, Shinya Yamanaka, to receive the Nobel Prize in Physiology and Medicine, just 6 years after its discovery, in 2012. The prize at that time was also shared with Sir John Gurdon who uncovered the mechanism of reprogramming in the late 1950s. These iPSCs were first produced from mouse fibroblasts by the transduction of four transcription factors, which when overexpressed, could completely change the fibroblast’s phenotype into that of an embryonic stem cell-like cell, capable of forming all cell types in the body, upon differentiation . Today, iPSCs are being produced from human cells and other species in many labs across the world, and production of these has been streamlined using more refined reprogramming techniques as well as different combinations of either genes, proteins, small molecules or miRNAs that can replace the function of the transgenes . What makes these cells so useful for studying disease is that they can easily be produced from patients suffering from the disease (patient-specific iPSCs) and be differentiated into the cell type/s affected by the disease, thus modulating and mimicking the disease in a dish.
The ability to produce patient-specific iPSCs has a number of advantages for both learning more about the disease itself and also in improving therapies and treatments. The ability to produce autologous stem cell populations from easy-to-access cells from the patient (e.g. blood cells and skin biopsies) overcomes the ethical conundrum of having to first produce a cloned human embryo using a donor cell from the patient and host de-nucleated oocyte and then having to destroy the cloned embryo, to harvest the pluripotent ESCs within . This in itself is an enormous breakthrough. There are several benefits in being able to have autologous cells from the patient. In patients that have degenerative diseases (e.g. diabetes, heart disease, osteoporosis, atherosclerosis and varying neurodegenerative diseases), the potential opportunity to have healthy cells transplanted back into the site affected is particularly appealing. The patient’s own cells can in fact also be corrected, in cases where genetic mutations induce the disease pathology. Alternately, autologous iPSCs derived from patients can also be used to improve the patient’s own medical treatment. In this case, the iPSC-derived cells can be screened in-vitro to determine which drugs prove most beneficial for the patients. This is one aspect of many approaches for developing tailored-specific treatments for patients, known as ‘personalized medicine’. The iPSCs, when differentiated into the target cells affected in the disease, can also be used to screen the potential new drugs being developed by Pharma, or potentially even used to discover new biomarkers of the disease. One of the latest developing fields in medical research includes the development of nanoparticles for treating disease, which are particularly attractive for use in brain diseases as they may pass easily through the blood-brain-barrier .
Despite the forefront in iPSC research, human ESC research is still in practice today for modelling neurodegenerative disease. Cell lines can be gene-targeted to induce familial-linked mutations, and in this way can be compared to genetically matched, unmodified control cell lines which are similar, if not more stringent controls than isogenic controls produced from iPSCs (see section on gene-editing below). Human ESCs are derived following the culture of the inner cell mass isolated from a pre-implantation embryo . Hundreds of lines have been produced over the years for research purposes for the study of cell pluripotency and regeneration, and these can be easily sourced from stem cell banks, registries or commercial companies. Generally, human ESCs are non-autologous, unless derived by somatic cell nuclear transfer from the patient. Only a handful of studies have produced autologous human ESCs from patients with disease [6, 7], and none exist at present for neurodegenerative diseases. Reasons for this are likely due to the ethical dilemmas in producing cloned human embryos and the technical challenges in cloned embryo production, compared to the ease in production of iPSCs.
Neurodegenerative diseases are characterized by progressive dysfunction of the nervous system as a result of loss of neuronal function in either the brain or the spinal cord and include Alzheimer’s disease (AD), frontotemporal dementia (FTD), Parkinson’s disease (PD), Huntington’s disease, amylotrophic lateral sclerosis and multiple sclerosis. Pluripotent stem cells (PSCs) have shown to be of particular promise for studying these diseases, since they can be expanded exponentially, hence providing much cell material for study. This is useful since it is particularly difficult to obtain tissue from the brain from patients suffering from the disease. In this review, we focus on the use of both iPSCs and PSCs in modelling AD, FTD and PD. In order for PSCs to deliver on their promises, it is important that clinical grade and safe cells can be produced for potential cell therapy. It is also important that these cells can modulate the disease accurately in the dish. That is, the cells must show the same pathology linked to the disease. In this review, we focus on how well iPSCs can model disease in a dish. We discuss how far the field has come in correcting the familial forms of AD, FTD and PD and how important the corrected mutations are for these diseases in relation to both the in-vitro studies and the potential for future cell therapy. Finally, we discuss what more is required to improve modelling in a dish, and where the current research is heading.
2. Use of gene-editing in modelling disease
Gene-editing involves insertion, deletion or replacement of DNA in the genome of an organism using engineered nucleases. This field has advanced considerably in just over a decade, thanks to the discovery and application of nucleases, combined with the latest molecular technology, which both enhance and improve the editing process. Gene-editing using designer nucleases was first applied to PSCs in 2007 . Since then, its application and use on PSCs have become widespread. It is currently being used by researchers to correct disease-causing mutations (endogenous gene correction) found within patient-specific iPSCs. In the case of PD, corrected autologous iPSCs through gene-editing are particularly promising for future cell transplantation studies, where diseased cells are genetically corrected and transplanted back into the patient’s brain. Another application for gene-editing in modelling disease is to produce genotypically matched control cell lines of disease iPSC lines. That is, because comparisons with age-matched healthy control lines from non-related persons are genetically different, which may impede on both phenotype and even differentiation capabilities . This use of gene-editing technology thus enables eloquent comparable studies in-vitro of disease phenotypes, which is directly linked to the mutation
The first generation of engineered nucleases produced were the zinc-finger nucleases (ZFNs), which were initially developed as chimeric restriction enzymes . These are modular proteins containing a
Another gene-editing tool is the transcription activator-like effector nucleases (TALENs). These are composed of a sequence-specific DNA-binding domain and a non-specific DNA cleavage module . The DNA binding domain contains a series of tandem repeats comprising 33–35 amino acids, similar to tandem repeats first discovered in the plant pathogen
The most recently developed method, which is even easier to use than TALENs and ZNFs is clustered, regularly interspaced, short palindromic repeats/Cas9-mediated genome-editing method (CRISPR/Cas9). This method consists of a specialized two-RNA structure containing CRISPR RNA (crRNA) and trans-activating RNA (tracrRNA), which are able to bind as a monomer to DNA strands next to a protospacer adjacent motif (PAM) composed of the sequence, 5'NGG'3 . This chimeric RNA is known as a guide RNA (gRNA) and facilitates a DSB by guiding an endonuclease (first derived from
To date, despite a surge in literature in gene-editing technologies, only three reports have been published that have led to the correction of iPSCs from patients with neurodegenerative diseases or alternately, insertion of disease-causing mutations into healthy PSCs. Two of these have been in the field of PD, and one in the field of FTD and all cases used ZFNs [24, 25] (Figure 1). In the case of PD, insertion of point mutations, A53T and G188A located in the gene, α-synuclein (
Together, these studies show that gene-editing is a particularly helpful tool for modelling diseases with iPSCs, as well as for helping to determine new molecular pathways involved in the disease by repair of the mutation and assessment for amelioration of the phenotype. It is likely that an increase in similar studies will soon emerge for PD and FTD as well as other neurodegenerative diseases, such as AD in the near future.
3. Current approaches to developing neural cells in a dish
Investigations on iPSC-derived neural cells can be performed either in two-dimensional (2D) models or three-dimensional (3D) models. Traditional stem cell research has been performed in 2D, predominantly by culturing cells that adhere to a plastic surface, and which form a flattened monolayer across the plastic surface. The advantage of this technique is that it is low cost and an easy system to use. Stem cells, however, can alternately be cultured in 3D. One popular 3D method is to culture cells in small spheres in suspension within the media, termed either ‘embryoid bodies’, or to differentiate them into neural progenitor cells that also cluster together in small spheres, termed ‘neurospheres’. These can be further differentiated into more mature neural cell types, which are often termed ‘engineered neural tissue’ or ‘organoid’ cultures. The cells can be cultured as described in aggregates, but can also be cultured in either the presence of microcarriers, on alginate microencapsulates, in thermoreversible hydrogel or in scaffolds . It is accepted today that neural stem cells (NSCs) isolated from primary tissue from foetal tissue or brain differ to neurospheres differentiated from EBs, as the former spheres tend to contain radial glial-like stem cells that are unable to form complex neural tissues such as the layered cortical neuroepithelium and complex pattern formations . In contrast, the stem cell-derived neurospheres can be instructed to form specific neural regions of the developing brain when exposed to potent mitogens/morphogens . Alternately, neural cells can also be cultured within artificially produced 3D scaffolds or in microwells formed within the plastic substrate that help to re-create a microenvironmental cue for the cells to form in 3D clusters. In fact, 3D cultures originated in the NSC field in the early 1990s when the first suspension cultures of rodent brain NSCs was performed , and have become a standardized way of culturing NSCs in-vitro in labs across the world. Today, there are several types of 3D scaffolds available, including metal, synthetic organic types made from polymers, synthetic inorganic materials, natural organic materials, natural inorganic material types and even nanostructure scaffolds . All of these have their advantages. For example, microcarrier systems allow for good diffusion properties and induce cells of high quality. Also, encapsulated cells in gels allow them to be protected from shear force-induced cell death, and thermoreversible hydrogel allows for rapid expansion of cells .
Comparative studies of 2D versus 3D cultures suggest that 3D culturing may improve the quality of the cell expression profile of the cultured cells as cells are influenced by the biochemical, mechanical and physical surface properties of the surrounding matrix in which they normally reside . One such comparative study showed neural-derived ESCs expressed more neural markers and greater neurite outgrowth when cultured in a 3D scaffold than the equivalent neural cells cultured in 2D . Furthermore, timing in differentiation appears to differ between 2D versus 3D cultures. In fact, stem cells appear to differentiate earlier in 2D culture when compared to culture in extracellular matrix gel or as spheres, shown by the earlier upregulation of differentiation markers . Whether this is abnormal or not has not yet been determined. Cell size and proliferation can also be altered by culture in 3D. One study has illustrated human ESCs cultured in 3D within microwells were smaller in size and divided more slowly compared to equivalent cells grown in 2D .
There has also been a recent surge in developing 3D models that better recapitulate the 3D complexity of the tissue in the body and which contain several cell types. The recent discovery that a human foetal-like brain could be recapitulated in the dish after culture of neural stem cells for several weeks was a remarkable discovery. This tissue was termed as a ‘cerebral organoid’ and was formed by embedding neural aggregates into Matrigel® droplets and culturing these in a bioreactor for 75 days . The tissue contained both early-born and late-born cortical neurons, suggesting more complex cortical neural development could be recapitulated in-vitro. It also contained interneurons, suggesting a mix of different progenitor origins were present in the tissue. Other researchers have also produced complex neural tissue with cortical layer patterning in 3D neural cultures, which depict both proliferative cell populations and post-mitotic cortical cell types; however, the complex stratification of the cortical layers has not yet been replicable . The addition of extracellular matrix molecules to both the substrate of 2D and within 3D culture systems may also be particularly advantageous for the growth and cellular expression of neural cell types, as shown by Lancaster and colleagues where neural aggregates were cultured in Matrigel® droplets .
There appears to be improvement in the cellular expression and cell function when cultured in 3D, as well as other physical and changes in size and growth. However, some drawbacks in using 3D scaffolds are the difficulties of performing molecular analyses on the tissue, which are related to problems in extracting the cells from the scaffolds or light refraction that emanate from the scaffold structures and which interfere with fluorescence microscopy. In addition, there are also seeding issues related to the complexity of some scaffold structures. Furthermore, 3D culturing is more labour-intensive and can also be difficult to scale up. Bioreactors help, in part, to solve this issue when cells are grown in spheres or small scaffolds and they also help through their spinning properties to distribute medium evenly throughout the culture. Use of bioreactors, however, requires extensive volumes of media, which can be costly when large volumes of cytokines or growth factors are required in the culture medium.
Despite the given advantages in use of 3D culture over 2D, disease modelling studies for AD, PD and FTD using PSCs have been performed using 2D cultures. One interesting research article relevant for AD demonstrates the advantages of producing 3D cultures. Choi and colleagues showed that transgenic human NSCs overexpressing either
4. Modelling Alzheimer’s disease using pluripotent stem cells
Alzheimer’s disease is the most prevalent type of dementia, which in most cases (approximately 90%) arises in patients with no known genetic link. However, some risk factor genes (e.g. presence of the allele ε4 of apolipoprotein E4 (
4.1. Sporadic AD with no known genetic mutations
The main pathology of sporadic AD is typical for the disease with the accumulation of toxic forms of Aβ peptides and tau protein. However, other proteins are also known to accumulate, including
4.2. Sporadic cases with mutations in APOE
The polymorphism of the
5. Familial AD
Pathology common to all familial mutations includes an earlier onset of the disease and increased amyloid plaque formation when compared to sporadic AD in many, but not all cases . Plaques tend to predominantly contain Aβ42 with often no increase in Aβ40 observed, contrasting that seen in sporadic AD . A summary of the PSC-derived neural cell pathology and comparative pathology known in familial AD patients is summarized in Table 1.
No increase in
|Yagi et al., 2011; Liu et al., 2014;|
Mahairaki et al., 2014
Duan et al., 2014
|D385N||Unknown||Decreased Aβ40||Koch et al., 2012|
Cotton wool plaques
et al., 2002
|Decreased Aβ40||Koch et al., 2012|
Atrophy of substantia nigra and cerebral cortex
Cotton wool plaques
|Ishikawa et al., 2005,84||Increased|
|Liu et al., 2014|
|Halliday et al., 2005||Increased|
|Liu et al., 2014|
Some cases with Lewy body pathology in frontal cortex and amydala
|Levy-Lahad et al., 1995; Rogaev|
et al., 1995
Jayadev et al., 2010
|Increased Aβ42:Aβ40||Yagi et al., 2011|
|duplication||Intercerebral haemorrhage, Diffuse brain atrophy|
Cerebral ventricular dilation, Intraneural Aβ40
Cerebellar purkinje cell atrophy
|Cabrejo et al.,|
Increased p-tau (Thr231)
|Israel et al., 2012|
Israel et al., 2012
|E693Δ||Early stages—limited brain atrophy and little|
accumulation of Aβ.
|Shimada et al., 2011||Decreased Aβ40 and Aβ42|
Elevated Aβ oligomers
|Kondo et al., 2013|
|Kondo et al., 2013|
Cortical and subcortical
|Lantos et al., 1992||Increased Aβ42:Aβ40|
Increased total tau
Increased p-tau (S262)
|Muratore et al., 2014|
5.1. PSEN1 mutations
Over 170 mutations in
The majority of iPSC lines modelling AD have in fact been produced from patients carrying mutations in
5.2. PSEN2 mutations
There are 23 known DNA variants reported in the
To date, only one study has investigated the pathology from neurons derived from a patient carrying the N141I mutation in
5.3. APP mutations
Three studies have investigated the pathology in neurons derived from patients carrying mutations in
6. Other models of AD
6.1. Trisomy 21
Trisomy 21 (also known as Down’s syndrome) results in the duplication of the APP gene, which is located on chromosome 21. People with this syndrome develop symptoms and pathology early in life, which are strikingly similar to AD . The extra copy of APP is considered the major factor in the AD-like symptoms and, in addition, duplication of Dyrk1A kinase (which is also located on chromosome 21), which phosphorylates tau, may also contribute to the pathology and symptoms . Increased Aβ peptides can be observed in early childhood which are the main candidate thought to induce the early onset of dementia . Since duplications of APP are observed in AD patients, trisomy 21 has also been used to model AD in many studies. One study has produced iPSCs from patients with trisomy 21 and neurons derived from the iPSCs showed perturbed Aβ processing, including increased Aβ40 and Aβ42 in long-term cultured neurons, as well as Aβ42 intracellular and extracellular aggregates . Furthermore, this study also reported increased p-tau and total tau, as well as increased cell death . This is the only study to date which reports cell death in an iPSC model of AD, which suggests this may be a relevant and worthy model of APP duplication and study of AD-like dementia.
To conclude, iPSCs from AD models tend to show early features of the disease in the dish, rather than distinct histopathological hallmarks (Figure 2). The most common observations include altered expression levels of Aβ and increased levels of tau. It might be that the main pathological hallmarks only develop after many years of protein aggregation and build-up in the cell.
7. Modelling frontotemporal dementia using pluripotent stem cells
Frontotemporal dementia accounts for a large proportion (50% of dementia cases that arise before the age of 60, and is the second most common early-onset dementia). This disease is characterized by the progressive loss and degeneration of the cortical neuron population, in the frontal and temporal lobes of the brain. Common symptoms include altered behaviour and deterioration in both speech and cognition [61, 62]. This disease has a much stronger genetic link than AD and PD. Approximately 40% of the cases are attributed to mutations in one of three genes, including microtubule-associated protein tau (
|expansion repeat||Brain atrophy|
Argyrophilic grain disease in limbic areas and orbital frontal cortex
Atrophy also in substantia nigra, brain stem and spinal cord
|Shinagawa et al., 2014|
DeJesus-Hernandez et al., 2011
|Almeida et al., 2013|
Almeida et al., 2013; Donnelly et al., 2013
Donnelly et al., 2013
|Hyperphosphorylated tau in DA neurons and glia and in brain|
stem and temporal cortex
Increased 4R tau isoform
|Ehrlich et al., 2015; Wren et al., 2015||Increased expression of 4R tau isoform, increased tau fragmentation|
Altered axonal mitochondrial transport
|Ehrlich et al., 2015: Iovino et al., 2015|
Ehrlich et al., 2015
Wren et al., 2015
Iovino et al., 2015
|Ex12 V337M FTDP-17-2||Frontotemporal atrophy|
Moderate parietal cortical
Atrophy of substantia nigra
|Domoto-Reilly et al., 2016||Increased tau fragmentation|
|Ehrlich et al., 2015|
Atrophy of substantia nigra
|Spillantini et al., 1998||Early maturation|
Altered axonal mitochondrial transport
|Iovino et al., 2015|
Caudate nucleus atrophy
Substantia nigra atrophy
Ubiquitin inclusions containing TDP-43
et al., 2007
Impaired WNT signalling
|Raitano et al., 2015|
|S116X||Unknown||Cellular stress||Almeida et al., 2012|
|Zhang et al., 2013|
et al., 2010
|Decreased survival||Bilican et al., 2012|
7.1. Sporadic FTD
Sporadic FTD has been modulated in-vitro by two independent studies to date [65, 66]. Brain atrophy is greater in the anterior cingulate compared to familial FTD cases . In one study, no characterization of the phenotype was performed. Another study also characterized patient iPSCs from a sporadic case of FTD and the cultured neurons showed greater cellular stress and oxidative stress compared to control cells . Whether all cases of sporadic FTD are in fact undiscovered genetic mutations is a distinct possibility, since there have been many recent discoveries of gene mutations lying behind the disease .
7.2. C90RF72 mutations
A GGGGCC repeat expansion in the noncoding region of
Two studies have produced patient-specific iPSCs from patients carrying this mutation. Characterization of the neurons derived from the iPSCs revealed they were more susceptible to cellular stress compared to control neurons . In addition, RNA foci were observed in the in-vitro produced neurons , but there is controversial evidence that suggests patient neurons have these . Both studies also showed cytoplasmic expression of RAN. Of interest was one other study that used patient-specific iPSCs to discover potential binding partners, e.g. ADARB2 to the expanded repeat region in an attempt to discover more about the mechanisms that lead to disease onset with this mutation  and to find out more about the gene’s actual function.
7.3. MAPT mutations
Two eloquent studies have generated iPSCs from patients carrying the N279K mutation and compared the in-vitro pathology directly to pathology in the deceased patient brain. Here, the cultured neurons had increased expression of the 4R tau isoform and fragmentation of tau , which helped to confirm the phenotype; however, neurite shortening, oxidative stress , cellular stress and enlarged vesicles  were also observed in the cultured neurons. The NFTs, however, were not able to be recapitulated in-vitro. Another study reported similar pathology in iPSC-derived neurons also carrying the N279K mutation . In addition, this same study also compared the N279K iPSCs to iPSCs carrying the
7.4. GRN mutations
Patients carrying mutations in
7.5. TARDBP mutations
Mutations in the
To date, only a fraction of the familial FTD mutations have been modelled using PSCs. Given the expanse of different mutations that exist as well as the broad pathology from each of the FTD variants, as well as within the variants themselves, it is important that the in-vitro studies can be correlated to the known pathology in the patients. Many different phenotypes can be observed in the dish, but the classical hallmarks appear to be missing in modelling the disease using iPSC-derived cells (Figure 3). How this might be improved upon is discussed more at the end of the chapter in the section under “Current limitations in modelling neurodegenerative disease using pluripotent stem cells”.
8. Modelling Parkinson’s disease using pluripotent stem cells
Parkinson’s disease is the second most common neurodegenerative disease, which is both sporadic and monogenic in form. The inherited monogenic form accounts for the minority of cases with approximately 5–10% of presented cases. The genetic contribution to Parkinson’s disease has been firmly characterized in the past few years to be directly induced by over 15 mutations in PARK loci, which are located within six genes:
The common idiopathic features of the disease are motor disturbances including resting tremor, rigidity and bradykinesia, as well as non-motor symptoms such as cognitive impairment, autonomic dysregulation, sleep deterioration and neuropyschiatric symptoms . These symptoms and disturbances arise due to the loss of nigrostriatal dopaminergic neurons in the substantia nigra pars compacta and the development of Lewy bodies in surviving neurons. This makes PD a particularly easy disease to modulate in a dish, as one predominant neuron type is affected. Despite this, there still lacks perfect differentiation protocols that result in the A9 type dopaminergic neuron in high proportion. Given the simplicity in the tissue affected by the disease, it has been considered that PSC-derived nigrostriatal dopaminergic neurons from healthy donors or genetically corrected iPSCs could be used for transplantation either into the striatum where they migrate to or in the substantia nigra where the cell bodies lie. In this case, many studies have attempted to improve the production and numbers of nigrostriatal dopaminergic neurons from PSCs . A summary of the PSC studies that model familial PD in a dish are shown in Table 3 along with known pathology in the patients.
|A53T (G209A)||Lewy body|
|Golbe et al., 1990||Oxidative stress|
|Ryan et al., 2013|
|A53T (G188A)||Soldner et al., 2011|
|Triplication||Lewy body pathology, hippocampal neuronal loss, temporal lobe vacuolation||Farrer et al., 2004;|
Muenter et al.,
|Devine et al., 2011|
Byers et al., 2011
Byers et al., 2011
|G2019S||Lewy bodies in|
Lewy body + AD pathology
Transitional Lewy body disease
SN neuronal loss,
tau pathology, +
SN neuronal loss
Lewy body pathology
|Giasson et al.,|
2006; Ross et al., 2006
Giasson et al
Ross et al., 2006
Rajput et al., 2006
Gaig et al., 2007
Gilks et al., 2005
Altered ERK signalling
Increased sensitivity to toxins
Increased expression of MAPT and p-tau
Mitochondrial DNA damage
|Nguyen et al., 2011|
Reinhardt et al., 2013
Reinhardt et al., 2013; Sanchez-Danes et al., 2012
Reinhardt et al., 2013; Cooper et al., 2012
Reinhardt et al., 2013
Sanders et al., 2014
Sanches-Danes et al., 2012
Fernandez-Santiago et al., 2015
|Not known||Increased sensitivity to toxins||Cooper et al., 2012|
|del_40bp ex3/del ex4+5||Not known||Oxidative stress||Aboud et al., 2012|
|del_40bp ex3||Lewy body|
|Farrer et al., 2001|
|Pramstaller et al., 2005|
|del ex4||SN neuronal loss, no Lewy bodies||Mori et al., 1998|
Hayashi et al.,
|del ex5||Only clinical features known||Proteosome dysfunction|
|Chang et al., 2016|
|Samaranch et al. 2010|
|Hedrich, et al.,|
|Oxidative stress||Seibler et al.,2011|
|Q456X homozygote||Increased sensitivity to toxins|
|Cooper et al., 2012|
|D525N/W577R compound||Cooper et al., 2012|
8.1. Sporadic PD
One study that produced iPSC lines from seven patients with idiopathic PD revealed that dopaminergic neurons produced in-vitro had reduced numbers of neurites, neurite arbourisation and increased autophagy . Another study observed methylation alterations in sporadic PD iPSC-derived dopaminergic neurons , which was the first to describe epigenetic dysregulation in the disease.
8.2. SNCA mutations
There are at least five mutations characterized within
Induced PSC lines have been derived from PD patients carrying either a triplication of
8.3. LRRK2 mutations
There have been five identified mutations in the
8.4. PARK2 mutations
8.5. PINK1 mutations
A mutation in
Together, the literature reveals a vast array of disease modelling studies using PSCs to model PD and a number of phenotypes have emerged in the dish although the classic hallmark, Lewy bodies and neurodegeneration, remain absent in the in-vitro cultures (Figure 4). Oxidative stress is a clear phenotype observed in many of the different studies and appears common for many of the mutations, but it is clear that the disease pathology is diverse and therefore the pathology in the iPSC-derived cells would also expect to be diverse.
9. Current limitations in modelling neurodegenerative disease using pluripotent stem cells
Modelling AD and FTD is definitely more challenging than modelling PD, since many different cell types and regions within the brain are affected by these diseases compared to PD. Therefore, these former diseases are considered less attractive for cell therapy compared to PD. In fact, there is much discussion in the scientific community on the embarkment of clinical trials for PD using human PSCs and it is therefore only a matter of time before these trials begin to emerge. However, the majority of the studies to date have been performed to understand more about the mechanisms of the disease and to find new targets that could be used for discovering new and more effective medicines for braking the disease and for improving symptoms and quality of the patient’s life.
One of the limitations observed to date in modelling the diseases is producing the right type of cells that are affected by the disease following differentiation of the iPSCs. In the case of AD and FTD, most studies have focused on evaluation of MAP2/TUJ1-positive neurons, cortical neurons and neural progenitors, which are often derived in heterogenic cultures. Whether these are the best cell types to examine in the dish is debatable. MAP2/TUJ1 expression is relatively unspecific and can label a vast number of different neuron subtypes, so it might be more important to use more specific antibodies to identify the specific neuron subtype that is being analysed. Cortical neurons are also numerous and some research, but not all, has identified the cortical subtype that has been produced in-vitro. In the case of AD, for example, the superficial cortical neurons are affected earlier on in the disease and the deeper cortical neurons are affected later . Therefore, knowing which cortical neuron subtype is produced in the dish will help to understand better the disease mechanisms and whether the pathology being observed is related to early or late stages of the disease. Some researchers are pursuing the development of more complex laminated cortical layer cultures in-vitro , which could be a great source of tissue for performing further studies on. The study of neural progenitors might be more relevant for looking at potential early mechanisms of the disease and to evaluate regions of the brain where progenitors exist, as well as the effect of the disease on the cell cycle. In the case of PD, protocols for the generation of dopaminergic neurons have been refined over recent years allowing for relatively high proportions of the correct ventral-mesencephalon type following differentiation . However, it is easy to forget the other cell types that can be affected in PD and the role they may also have in the disease. Therefore, the identity of the cell types produced needs to be more carefully defined in studies to help reveal more details about how the disease affects that particular cell type. More complex in-vitro models could also help to mimic the in-vivo environment better, which might help to reveal more phenotypes associated with the disease.
It is clear from the studies that the phenotypes, and in particular, the classic hallmark pathologies, are not represented in-vitro. The reasons for this are not really clear, but may relate to the fact that neural subtypes may be relatively young in the dish compared to the neural cells found in the patients. The evidence so far reveals that the iPSC-derived cells can model early changes related to the disease and therefore might prove useful for finding ways to reverse the disease or slow it down in the early or pre-symptomatic stages. If we are to evaluate some of the classic hallmarks, which are generally missing in culture (i.e. neurodegeneration, amyloid plaques, NFTs and Lewy bodies), then it might be needed to accelerate the aging of neurons in-vitro using artificial methods. One approach successfully used to age neurons in a dish was demonstrated using iPSCs from PD patients . Overexpression of progerin (a gene linked to a disease of accelerated aging, progeria) was performed, which resulted in a pronounced PD phenotype of the dopaminergic neurons. The neurons were able to show much more pathology compared to non-aged neurons, including dendrite degeneration, loss of tyrosine hydroxylase expression (a typical marker of dopaminergic neurons) and Lewy-body-precursor inclusions within the neurons. This has not yet been applied to AD or FTD, but is an intriguing tool that could help to elaborate more of the classic pathologies/phenotypes in the dish.
To conclude, disease modelling of neurodegenerative diseases using PSCs has developed dramatically over a short period of time (a space of about 5 years). Already new mechanisms related to AD, FTD and PD have been discovered and these will likely lead to the development and trial of new medicines for the disease. There are many reported phenotypes that have been linked to the disease that can be reversed when familial mutations are genetically corrected using gene-editing technology. However, not all phenotypes have been reported so far, which may be linked to the cell types evaluated, the relatively simple systems used and also the relatively young neural cell types analysed. New studies developing 3D cultures and more complex tissue types may help move the field forward. In addition, new technologies that accelerate aging in the dish are also likely to help overcome these limitations. Thus, iPSCs have already been useful for uncovering some of the mysteries surrounding neurodegenerative disease and the future will likely lead to uncovering more about the disease mechanisms and how we can repair and treat the dysfunctioning cells before they are lost in the patient.
The author’s current research and activities are currently funded from the People Programme (Marie Curie Actions) of the European Union Seventh Framework Program FP7/2007-2013/ under REA grant agreement n°PIAPP-GA-2012-324451 (STEMMAD), as well as the Innovation Fund Denmark supported project, BrainStem (a Stem Cell Center of Excellence in Neurology).