Similar to our in vitro results

Similar to our in vitro results, transplanted iNSCs exhibited in vivo-characteristics, transcriptome profiles, neural and metabolic programs similar to those of transplanted NSCs. In contrast to engrafted forebrain NSCs, however, transplanted iNSCs strongly expressed caudal regional marker genes and only marginally acquired rostral gene expression. Thus, iNSCs vastly retained their caudal positional identity in vivo suggesting regional stability and a predominantly cell-autonomous regulation of positional marks in transplanted cells in a forebrain microenvironment. These findings are in line with observed limited plasticity of iNSCs in establishing rostral domains in vitro and indicated that rostral cell patterning of iNSCs most likely requires strong guidance cues and even employment of rostral transcription factors such as Foxg1 (Lujan et al., 2012) or Wnt7b during earlier phases of iNSC generation before the establishment of a committed iNSC stage in vitro. It should be noted though that improper positional programs in transplanted iNSCs did not affect their integrity or the migration of endogenous neuroblasts from the SVZ. It remains to be shown how iNSCs with either rostral or caudal positional marks influence outcomes after transplantation into forebrain domains in animal models of neurological disease and injury. Furthermore, it would be important to determine the mechanisms, by which iNSCs acquire neural, positional programs during neural conversion. These studies would not only advance the field of direct reprogramming in vitro but could also provide important data for the emerging field of transcription factor-mediated neural reprogramming in vivo. The following are the supplementary data related to this article.
Accession numbers The data discussed in this publication have been deposited in NCBI\'s Gene Expression Omnibus and are accessible through GEO Series accession number GSE73722 (http://www.ncbi.nlm.nih.gov.eleen.top/geo/query/acc.cgi?token=ydyrsqoyttaztqb&acc=GSE73722).
Author contributions
Acknowledgements We would like to thank Ingrid Gelker, Martina Sinn, Martin Stehling, Elke Hoffmann and Claudia Kemming for outstanding technical assistance. This study was supported by research funding from the IMF at University Hospital Münster to GH (I-HA-111219) and from the DFG to TK (SFB-TRR128-B7). The authors declare no conflict of interest.
Introduction Genomic imprinting is an epigenetic mechanism that ensures the differential expression of imprinted genes in a parent-of-origin fashion. The (-)-p-Bromotetramisole Oxalate of imprinted genes are marked with their parental origin by DNA methylation at differentially methylated regions (DMRs), which are CpG-rich-cis-elements within the locus (Reik and Walter, 2001). In order to be inherited from one generation to the next, these epigenetic marks are erased early in life in primordial germ cells and reset in the germline as per sex (Hajkova et al., 2002). The altered methylation of imprinted genes leads to improper gene dosage during embryonic development and has been associated with several pathologies, including cancers and neurological disorders (Reed and Leff, 1994; Orstavik, 1999; Feinberg, 2004; Demars and Gicquel, 2012; Brioude et al., 2013; McCann et al., 1996; Takai et al., 2001). Recent studies have suggested that assisted reproductive technologies (ARTs), such as superovulation, in vitro fertilisation and embryo culture, favour acquisition of imprinting errors, which can lead to diseases and developmental defects (DeBaun et al., 2003; Gicquel et al., 2003; Maher et al., 2003; Orstavik et al., 2003; Borghol et al., 2006; Bowdin et al., 2007; Khoueiry et al., 2008; Grace and Sinclair, 2009; Chen et al., 2010; Ibala-Romdhane et al., 2011; Khoueiry et al., 2013). During the early stages of embryonic stem cells (ESCs) isolation from pre-implantation stage embryos, embryonic cells are subject to intense in vitro manipulation and environmental changes that may impact the epigenetic status and irreversibly alter the capacity to generate ESC lines or to exhibit the full differentiation potential of genuine ESCs. Primate ESCs often show altered DNA methylation on imprinted genes, particularly imprinted genes, such as H19/insulin-like growth factor2 (IGF2) (Fujimoto et al., 2006; Mitalipov, 2006; Mitalipov et al., 2007; Frost et al., 2011). However, it is unknown whether these alterations emerge during ESC isolation and whether they are correlated with the ESC outcome. To address these questions, we analysed the methylation profiles of two well-characterised DMRs, H19/IGF2 and SNRPN DMRs, while deriving monkey ESCs. The H19/IGF2 DMR is the best candidate for this study because its methylation status is particularly sensitive to changes in culture conditions and differentiation (Sasaki et al., 1995; Doherty et al., 2000; Khosla et al., 2001; Mann et al., 2004). The H19/IGF2 DMR acquires methylation in the paternal germline and is characteristically unmethylated on maternal alleles. The H19/IGF2 DMR regulates the expression of two oppositely imprinted genes, such as IGF2 and H19. The IGF2 locus encodes IGF2, an autocrine/paracrine mitogen, and transcription of H19 produces a non-coding RNA, which is a precursor of a microRNA called miR-675 that negatively affects cell proliferation (Keniry et al., 2012). Hyper-methylation of this DMR can result in IGF2 overexpression and is linked to an increased frequency of Beckwith–Wiedemann syndrome (DeBaun et al., 2003; Weksberg et al., 2003), whereas hypo-methylation of the paternal allele is associated with the Silver–Russell syndrome, which is characterised by slow growth before and after birth (Gicquel et al., 2005). In contrast to the H19/IGF2 DMR, the SNRPN DMR is methylated on the maternal allele and unmethylated on the paternal allele. In humans, it is located on chromosome 15q11–13, which is a region involved in Prader–Willi and Angelman syndromes (AS) (Reed and Leff, 1994; Leff et al., 1992; Buiting et al., 1995). Methylation of this DMR is not sensitive to environmental alterations, including the in vitro manipulation of mouse ESCs (Schumacher and Doerfler, 2004), which makes it a good marker of methylation stability.