The LARGE Principle in AFiPSCs when compared to the undifferentiated cells (scale bars = 20 mm). (B) qRT-PCR and gene expression profiling (microarray) revealed down-regulation of pluripotency markers POU5F1 and NANOG, but up-regulation of trophoblast markers CDX2, KRT7, HAND1, FOXF1, GATA3 and ID2 upon BMP2- or BMP4-treatment of AFiPSCs. Data are presented as LOG2 ratios (BMP-treated versus untreated AFiPSCs) and standard deviation. (C) Immunofluorescence-based detection of the placental hormone human chorionic gonadotropin (hCG) in AFiPSCs after treatment with 10 ng/ml BMP4 and 10 mM SB431542 over a period of seven days (scale bars = 200 mm). doi:10.1371/journal.pone.0013703.g003 NF2, CD59, TNFSF10, NT5E) and an iPSC (AFiPSC)-specific gene expression signature (555 genes, e.g. CNTFR, SIX6) (Figure 4C, the entire gene lists are presented in Table S3). Expression of senescence-associated genes in primary AFCs and AFiPSCs To investigate the effect of reprogramming on bypassing senescence observed in primary AFC cultures (Figure 1A-II, - III), we analyzed the expression of senescence and telomere- associated genes in young primary AFCs (P6) and senescent AFC (P17) compared to AFiPSC lines (approximately P20). From a list of 116 senescence-associated genes (Table S4) derived from the Gene Ontology database [35], including those described by Vaziri et al. [45], we identified 64 genes as significantly differentially expressed in AFCs at passage 17 compared to the union of all AFiPSC lines (Figure 5). Of these, telomere-associated genes and genes involved in regulating the cell cycle, e.g. MAD2L2, PARP1, RPA3, DKC1, MSH6, CHEK1, PLK1, POU2F1, CDC2, BLM, WRN, DNMT1, DNMT3B, LMNB1, and CDT1, were down-regulated in primary AFCs compared to AFiPSCs and ESCs. In contrast, PIN1, LMNA, GADD45A, CBX6, NOX4, ENG, HIST2H2BE, CDKN2A, CDKN1A, GDF15 and SERPINE1, among others, were up-regulated in primary AFCs compared to AFiPSCs and ESCs. Activation of a common ESC-like core transcriptional regulatory network in AFiPSCs and FiPSCs In order to narrow down the self-renewal and pluripotency signature gene list obtained by comparing global gene expression patterns of AFCs, AFiPSCs and ESCs (1299 genes, Figure 4C), we compared the same ESC samples with FiPSCs and the respective parental fibroblast line HFF1 (Fibs) (Figure 6A, the entire gene lists are presented in Table S5) [31]. Using the resulting equivalent self- renewal and pluripotency gene signature, we could detect the overlap between the two self-renewal and pluripotency gene lists derived from the separate analyses (AFiPSCs/ESCs: 1299 genes in the self-renewal/pluripotency signature, FiPSCs/ESCs: 922 genes in the self-renewal/pluripotency signature). This revealed 525 genes expressed in common in all our pluripotent cell types (AFiPSCs, FiPSCs and ESCs), highlighting their role in maintain- ing self-renewal and pluripotency (Figure 6B, the corresponding gene list is presented in Table S6). To gain further insight into the gene regulatory network (GRN) that induces and maintains pluripotency in AFiPSCs and FiPSCs and to define distinct functions of the 525 core self-renewal-associated genes in the undifferentiated embryonic stem cell state, we identified the overlap of these 525 genes with the list of genomic regions bound by either OCT4 alone or by OCT4, SOX2 and NANOG as identified in human ESCs by ChIP-on-chip analyses [46,47]. This, in turn, revealed a subset of genes expressed in all of our pluripotent cell lines, which are part of an ESC-specific transcriptional regulatory network, including, for example, POU5F1, SOX2, NANOG, DPPA4, LEFTY2 and CDH1 (Figure 6C). To emphasize the established role of all the heatmap-listed genes in the regulation of the tightly controlled balance between the undifferentiated, self-renewing, pluripotent versus the differentiated ESC state, we combined the heatmap data in Figure 6C with gene expression data derived from siRNA- mediated OCT4 knockdown in ESCs [39]. The LARGE Principle of Cellular Reprogramming What can be gleaned from the global gene expression analyses presented here but also from other iPSC-based transcriptome analyses [48,49], is that induction of pluripotency is associated with the transcriptomes of the parental cells shifting towards a distinct ESC-like state, irrespective of the cell source. More precisely, for a distinct set of genes, which are expressed in the parental cell line, expression is lost (L), whereas the expression of another group of genes is acquired (A) in the process of iPSC generation. In turn, the expression of a third set of genes, detectable in the parental cells, is retained (R) in the corresponding iPSCs. We refer to this as the LARGE (Lost, Acquired, Retained Gene Expression) Principle of Cellular Reprogramming. Also referring to other studies [48–50], we propose that these particular LARGE patterns are the key to understanding similarities and differences between iPSCs and ESCs and their parental cell lines on the one hand as well the heterogeneity of different iPSC types on the other. As transcription factors normally influence gene expression of several downstream targets and, thus, are likely to play a fundamental role in this concept, we used gene expression patterns of transcription factors to illustrate the LARGE concept. For this purpose, we made use of the data from the Venn diagram analyses of AFCs/AFiPSCs/ESCs and Fibs/FiPSCs/ESCs (Figures 4A and 6A, Tables S3 and S5). For each of the Lost (genes expressed in donor cells, but not in iPSCs), Acquired (genes expressed in iPSCs, but not in the donor cells) and Retained (genes expressed simultaneously in donor cells and iPSCs, excluding genes of the house keeping gene signature) Gene Expression sets, we arbitrarily picked out genes associated with the Gene Ontology term for transcription factor activity (GO0003700) [35]. Of these, the 12 transcription factors with the lowest (Lost), highest (Acquired) or least varying (Retained) expression change, when comparing AFiPSCs or FiPSCs with the corresponding parental cells, are depicted in the heatmaps in Figure 7. As a result, the group of lost transcription factor gene expressions included, for example, HOXB7, HOXA9, HOXA10, PAX8, DSCR1, MYC in AFiPSCs and EMX2, FOXF2, FOXF1, MYC, KLF4 in FiPSCs. The acquired gene expression set can be further divided into two groups on the basis of present or absent overlaps between the two analyses for AFiPSCs and FiPSCs: those, which are universally acquired self-renewal genes present in both, AFiPSCs and FiPSCs, or, more generally, in all pluripotent iPSCs (e.g. POU5F1, SOX2, NANOG), and those acquired gene expressions, which are rather iPSC type-dependent (e.g. SIX6, EGR2 (AFiPSCs) or PKNOX2, HOXD4, HOXD10 (FiPSCs); DLX5 (AFiPSCs & FiPSCs)). The retained gene expression sets included genes like PKNOX2 (AFiPSCs); HMBOX1, MGA (FiPSCs) or RAXL1 (AFiPSCs & FiPSCs). Discussion Ground state pluripotency of AFiPSCs We have shown that cellular reprogramming of primary AFCs results in a fully pluripotent iPSC type, which is in line with recent PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13703