The LARGE Principle in AFiPSCs Figure 7. The LARGE Principle of Cellular Reprogramming. (A) To illustrate the concept of LARGE (Lost, Acquired, Retained Gene Expression), the top 12 genes with transcription factor activity and either low (LOST), high (ACQUIRED) or unchanged (RETAINED) expression values of iPSCs relative to the parental cell line were selected from the various gene expression signatures of AFCs, AFiPSCs, ESCs, Fibs and FiPSCs (Figures 4C and 6A). Significantly up-regulated or down-regulated gene expression in AFiPSCs compared to AFCs or FiPSCs compared to Fibs is indicated by asterisks (*) FDR-adjusted P-Value ,0.05, **) FDR-adjusted P-Value ,0.01, ***) FDR-adjusted P-Value ,0.001). (B) Schematic diagram of the LARGE Principle of Cellular Reprogramming. doi:10.1371/journal.pone.0013703.g007 AFiPSCs in basic and applied research, although it could be argued that the teratoma formation ability acquired by AFiPSCs hampers their use in cell replacement therapies. Yet, this is a feature of all kinds of iPSCs and ESCs, which still hold a lot of promise in this respect. Presumably, ways will be found to exploit the full differentiation potential of iPSCs while circumventing tumor formation risks, for instance, by developing accurate strategies to sort out differentiated cells of interest from potential tumorigenic stem cells. Cellular reprogramming bypasses senescence of bulk primary AFCs One of the great advantages of AFiPSCs over their bulk primary counterparts for any desirable application is their acquisition of the ability to propagate indefinitely. The data presented herein suggest, that this phenotypically rejuvenated appearance of AFiPSCs is based on a gene regulatory network, which averts or at least markedly delays the onset of senescence. This is based on the fact that primary AFCs and AFiPSCs and ESCs exhibit opposing expression patterns related to a large number of senescence-associated genes. In particular, we could detect high expression levels of various cell cycle and telomere elongation- associated genes, such as MAD2L2, PARP1, RPA3, DKC1, MSH6, CHEK1, PLK1, POU2F1, CDC2, LMNB1 and CDT1, as well as TERT itself in AFiPSCs in contrast to primary AFCs. The p53/ p21 pathway plays a pivotal role in inducing and maintaining senescence [51]. Accordingly, mRNA levels of several p53 target genes, which are known to be up-regulated in senescent cells [52– 54], e.g. CDKN1A (p21), GDF15, and SERPINE1, were strikingly elevated in primary AFCs compared to AFiPSCs and ESCs. In contrast, low level gene expression of DNMT1 and DNMT3B were detected in bulk primary AFCs, whereas these genes are significantly up-regulated in AFiPSCs. Besides their function in establishing and maintaining CpG methylation patterns during embryonal development, they are also known to repress CDKN1A transcription in opposition to and potentially independent of p53 [54,55]. Hence, it could be anticipated that high expression levels the DNMTs may repress CDKN1A and, thus, senescence in AFiPSCs. Taken together, there is evidence that senescence is bypassed upon the activation of a self-renewal and pluripotency program in reprogrammed AFCs, which is in line with our previous findings [31]. However, further studies are needed to assess the actual ability of AFiPSCs to restore telomere restriction fragment length to an ESC level, a subject of controversial discussion in the iPSC field [45,56,57]. The LARGE Principle of Cellular Reprogramming and ESC- specific gene expression signatures Mechanistic and functional aspects of cellular reprogramming in general, and of AFCs in particular, can be highlighted on the basis of the presented comparative transcriptome analyses of AFiPSCs, ESCs (H1, H9) and FiPSCs and the corresponding parental cell lines. For the different iPSC types we have identified genes, the expression of which are either lost (L), acquired (A) or retained (R) upon the induction of pluripotency. We refer to this as the LARGE (Lost, Acquired and Retained Gene Expression) Principle of Cellular Reprogramming. Some of these gene expression patterns, including several signature genes, are discussed. The donor cell (AFC)-specific gene signature contains putative immune-suppressive factors, such as CD59, TNFSF10, and NT5E (CD73) [58–60], which are likely to contribute to the immune- privileged characteristics of primary AFCs [14]. Their expression is lost upon reprogramming. Whether this affects potential therapeutic applications of AFiPSCs compared to primary AFCs remains to be elucidated. Interestingly, active gene expression, which is lost upon reprogramming, could also be observed for MYC (AFiPSCs & FiPSCs) and KLF4 (FiPSCs). This supports the idea that the main function of KLF4 and c-MYC in the process of reprogramming is to accelerate or enhance the efficiency by increasing a balanced cellular proliferation, while in pluripotent cells they seem to be dispensable [61–63]. Among the expressed genes, which are universally acquired during reprogramming processes, independent of the cell source, are key pluripotency-regulating factors, such as POU5F1, SOX2 and NANOG. These establish a core gene regulatory network essential for maintaining self-renewal and pluripotency [46]. Yet, at the same time, expression of genes known to be involved in differentiation and development, such as EOMES and HAND1, are acquired [39,47,64]. These genes are direct targets of OCT4, SOX2 and NANOG [46]. They are up-regulated upon OCT4 knockdown, and, therefore, negatively regulated by OCT4 [39]. Yet, we have observed low level expression of some developmen- tal-related genes in all our pluripotent cell types and also in repositories of ESC and iPSC-related microarray data (e.g. http:// amazonia.transcriptome.eu/temp/histo_7ebe096c97857d933b94c d30a6a120cf.png) [65]. It is conceivable, that this observation is an artefact of certain cell culture conditions or of spontaneously differentiating cells present in iPSC cultures. If, however, these genes are indeed expressed at moderately low levels in pluripotent cells, it would be worth investigating, whether this is due to distinct epigenetic marks on the promoters of these genes, similar to the concept of bivalent chromatin structures, which mark poised stem cell genes [66]. Furthermore, amongst the expressed genes, which are acquired in a cell type-dependent manner during cellular reprogramming, are those implicated in developmental processes, for example, SIX6, EGR2, HOXD4, HOXD10, PKNOX2 and DLX5 [67–71]. Likewise, the list of Retained genes in both, AFiPSCs and FiPSCs, included transcription factors, such as RAXL1, which is also involved in the regulation of developmental processes [72]. Persistent gene expression has already been reported to contribute to differences between various iPSC types and ESCs [48] and may result in variable differentiation behaviours of iPSCs, regardless of which reprogramming technique was applied [50]. Therefore, the impact of such active, developmental genes on the ability to maintain the pluripotent state and on directed differentiation processes of different iPSC types deserves further investigation. It is tempting to speculate, that the expression of some of the distinct signature genes are due to viral integrations into the target cell’s genome. However, since these signature gene expressions were identified as a result of a group-wise analysis of several AFiPSCs/ FiPSCs lines versus the parental cell lines and ESCs, they are unlikely to be attributable to clone-specific viral integrations in most cases. A PLoS ONE | www.plosone.org 12 October 2010 | Volume 5 | Issue 10 | e13703