In view of their potential use in cell-based therapy for patients with PD, midbrain DA neurons were one of the first cell types to be generated from iPSCs. The resemblance between iPSC-derived DA neurons and true DA neurons has been studied based on a number of mainly morphological and functional properties, as well as mdDA-specific gene sets (Hedlund et al., 2008; Kriks et al., 2011; Wernig et al., 2008). However, the risk of tumor growth and the heterogeneous molecular backgrounds of embryonic stem cell (ESC)- and iPSC-derived DA neurons have precluded the use of these prostaglandin receptor for therapy in humans (Momčilović et al., 2014; Salti et al., 2013). Gaining a detailed understanding of the genetic and epigenetic signatures of ESC- and iPSC-derived DA neurons is therefore a critical step toward establishing cell-based therapy as a viable treatment for PD. In this study, we present a comparative genome-wide profile of the genetic and epigenetic features of iPSC-derived DA neurons and their primary counterparts. Although iPSC-derived DA neurons showed characteristics that are widely used to classify functional mdDA neurons (Ganat et al., 2012; Kriks et al., 2011; Sundberg et al., 2013), caused a reduction in rotation behavior in a rat model for PD, and adopted many of the genetic and epigenetic features of their primary counterparts, we also observed major deviations that may interfere with proper functionality after grafting.
Since DA neurons obtained via induced pluripotency undergo defined developmental stages in vitro, we compared them with primary embryonic and postnatal mdDA neurons. To generate iPSCs and differentiate them into DA neurons, we applied a combination of currently widely accepted and standardized protocols (Chambers et al., 2009; Kawasaki et al., 2000; Takahashi and Yamanaka, 2006). We made use of transgenic Pitx3 mice (Maxwell et al., 2005) to isolate primary mdDA neurons and reprogram embryonic fibroblasts into iPSCs, which were subsequently differentiated into DA neurons. The transcription factor PITX3 has been demonstrated to be one of the most stringent markers for fully differentiated, functional mdDA neurons (Smidt et al., 2003, 2004), and expression of the PITX3-GFP reporter allowed us to strictly identify and FACS purify iPSC-derived and primary mdDA neurons.
Although transcript analysis indicated that proviral gene expression introduced during reprogramming was silenced in iPSC-derived mdDA neurons, we still found residual expression of fibroblast markers. It was previously shown that iPSCs are prone to differentiate along their somatic parental lineages because they maintain a parental epigenetic memory (Bar-Nur et al., 2011; Kim et al., 2010; Sullivan et al., 2010). Although some studies suggested that such epigenetic memory is restricted to early-passage iPSCs (Polo et al., 2010), others reported that parental epigenetic states also persist in late-passage iPSCs (Kim et al., 2011). This could explain why we observed fibroblast marker expression and a permissive DNA methylation state for some of the fibroblast genes in PITX3-GFP-positive cells derived from high-passage (p15–p20) iPSCs.
To determine whether the epigenetic states of the analyzed neuron populations reflected similarities and differences in gene-expression profiles, we analyzed global DNA methylation patterns in primary embryonic mdDA neurons (E14.5) and iPSC-derived DA neurons. In contrast to the case with undifferentiated iPSCs, we found strong general de novo methylation in iPSC-derived DA neurons. Earlier studies reported that PSCs mostly contain methylation-free promoters, as well as methylation-free intergenic and orphan CGIs (Illingworth et al., 2010). De novo methylation was only found upon loss of pluripotency, suggesting that transcriptional repression in PSCs is established predominantly via other mechanisms (Fouse et al., 2008; Mohn et al., 2008). Indeed, we observed that most de novo methylation events occurred during differentiation from pluripotent cells to multipotent precursors isolated from an iPSC-derived NESTIN-GFP reporter line (data not shown).