In this study, we describe the generation and characterization of a collection of iPSC lines representing age- and sex-matched patients of sporadic and LRRK2-associated PD, as well as control individuals. Consistent with previous studies describing the generation of patient-specific iPSC in the context of PD (Park et al, 2008a
; Soldner et al, 2009
) or other conditions (Dimos et al, 2008
; Park et al, 2008a
; Raya et al, 2010
), the efficiency of iPSC generation varied among different individuals, but did not depend on the presence or type of disease, nor on the donor age. We chose retroviral delivery of reprogramming transgenes to secure the derivation of iPSC lines from valuable biopsy material, because of the higher efficiency of this system in our hands compared to that of lentivirus-based systems. A clear advantage of the latter is the possibility of using inducible excisable lentivirus (Soldner et al, 2009
), so that iPSC are free of reprogramming transgenes. However, we reasoned that a careful analysis of the silencing of integrated transgenes in our iPSC lines, together with the generation of independent iPSC lines from several patients representing each condition, would rule out any confounding effects due to residual transgene expression in individual iPSC lines, while still allowing a high efficiency of iPSC generation. In any case, we omitted c-MYC from the reprogramming cocktail due to the possibility of its interaction with LRRK2 in the context of eukaryotic initiation factor 4E (eIF4E) function regulation (Imai et al, 2008
; Ruggero et al, 2004
All the iPSC lines tested in our study were competent to give rise to DAn, although the efficiency at which they did so varied among lines. Similar findings have been reported for iPSC in a variety of differentiation protocols, including neuronal lineages other than DAn (Hu et al, 2010
), cardiomyocytes (Zhang et al, 2009
) and haematopoietic cells (Woods et al, 2011
). Importantly, the variability in differentiation efficiency of our iPSC lines was independent of the presence or type of disease, indicating inter-line variation, rather than a result of the disease. Moreover, DAn differentiated from Ctrl- or PD-iPSC appeared morphologically and phenotypically indistinguishable after 30 days in culture, in agreement with a previous report that analysed the DAn differentiation ability of IP-PD iPSC (Soldner et al, 2009
), and extending these findings to iPSC derived from LRRK2-PD patients. Despite this similarity, we found anomalous accumulation of SNCA in DAn differentiated from LRRK2-PD iPSC, compared to those from Ctrl- or ID-PD iPSC. The identification of this PD-related phenotype in LRRK2 mutant DAn is consistent with the notion that LRRK2 and SNCA may participate in intersecting pathways (reviewed in Cookson, 2010
), and with the fact that LRRK2 can accelerate mutant SNCA-induced neuropathology in mice in a dose-dependent manner (Lin et al, 2009
). Moreover, these results demonstrate the ability of iPSC-based cellular systems to recapitulate PD-related pathology and to model a monogenic form of PD.
Probably the most significant finding of our work is the identification of spontaneous phenotypes in long-term cultures of DAn from both idiopathic and LRRK2-associated PD. To our knowledge, this is the first time such phenotypes have been described. Indeed, Soldner and colleagues did not find any differences between DAn differentiated from ID-PD iPSC and from control iPSC (Soldner et al, 2009
), a fact that they attributed to the short time span of cultured neurons (32–42 days). Moreover, these authors suggested that additional manipulations might be necessary to accelerate PD-pathology related phenotypes in iPSC-derived DAn in vitro
, such as increasing oxidative stress, challenge with neurotoxins or overexpressing PD-related genes (Soldner et al, 2009
). Thus, it may not appear entirely unexpected that DAn differentiated from our LRRK2-PD iPSC displayed PD-related alterations in vitro
. However, during the writing of this manuscript, a study was published that found no alterations in DAn differentiated from iPSC from one LRRK2-PD patient, unless these were challenged with H2
, 6-hydroxydopamine (a PD-related neurotoxin) or MG-132 (proteasome inhibitor; Nguyen et al, 2011
). We believe that the lack of spontaneous PD pathology-related phenotypes in previous iPSC-based models of ID-PD (Soldner et al, 2009
) and LRRK2-PD (Nguyen et al, 2011
), in contrast with our results presented here, may be due to at least two reasons: First, the longer time span of cultured neurons (up to 75 days) in our experiments, which may have induced culture-related stress conditions mimicking in vivo
aging in PD patients, and thus accelerated the development of PD-related phenotypes in vitro
. In support of this, we found that DAn differentiated for 30 days from ID-PD- or LRRK2-PD iPSC, a time when no alterations (other than SNCA accumulation in LRRK2-PD iPSC-derived DAn) were evident, showed increased susceptibility to sub-lethal concentrations of the PD-related toxin MPP+ (Fig S11
of Supporting information). Thus, it is likely that the prolonged culture of these cells in our 75-day differentiation protocol served to amplify the intrinsic susceptibility of PD-iPSC-derived DAn to undergo neurodegeneration in response to aging, the most important PD-related susceptibility factor. Second, it should be noted that the majority of iPSC-derived neurons in our experiments were of vmDA neuron phenotype, whereas, this cell population was much rarer in previous studies, with Soldner and colleagues reporting ~10% of DAn (TH+/TUJ1+ cells) of a phenotype that was not examined in detail (Soldner et al, 2009
), and Nguyen and colleagues reporting less than 1% of neurons of a vmDA phenotype (Nguyen et al, 2011
). We believe that the enrichment of vmDA neuron in our experiments facilitated the identification of PD pathology-related phenotypes, which are known to affect preferentially this specific subtype of DAn.
Our iPSC-based PD model should help investigating the pathogenic mechanisms that underlie PD neurodegeneration. Thus, although several lines of evidence suggest a causal link between impaired autophagy and the development of PD-related pathology, definitive proof for this has been elusive (reviewed in Yang & Mao, 2010
). Our findings of defective autophagosome clearance in DAn from PD patient-specific iPSC, together with the positive correlation between the expansion of the autophagic compartment and the degree of morphological alterations in these cells, provide strong support for this hypothesis. In addition, the iPSC-based PD model described here should also prove an invaluable tool to investigate early functional alterations that predate the onset of neurodegeneration, which surely will help identifying potentially new therapeutic targets for the prevention, rather than rescue, of PD-related neurodegeneration.
Overall, our studies demonstrate the potential of iPSC-based technology to experimentally model the pathogenic mechanisms of late-onset diseases such as PD. Not only did we find PD pathology related phenotypes in long-term cultures of DAn representing a monogenic form of PD, but also in those from patients of ID-PD. This critical point indicates that the cause for the increased susceptibility of ID-PD-derived DAn to undergo degeneration in vitro after long-time culture, albeit complex, is encoded in the genome of ID-PD patients, or at least of those tested in our study. Therefore, intrinsic cell-autonomous factors, rather than environmental influences, appear to be sufficient to trigger neurodegeneration of DAn from PD patients, in a process that requires time to manifest itself, but that can be modelled within the time-frame of in vitro experiments. We believe that key aspects to the success of our strategy were the efficient differentiation of the PD relevant cell type, the ability to maintain DAn cultures over a long-term culture span and the use of multiple patients per condition, which allowed controlling the inherent variability of human pluripotent stem cell lines. In this way, we have identified in vitro phenotypes associated with ID and/or LRRK2 PD, which could be harnessed as readouts for drug screening studies. Our findings provide important conceptual and technical advances for the understanding of PD pathogenesis and, eventually, for the identification of novel therapeutic strategies in this disease.