probable explanation could be that these gene expression patterns are due to an incomplete erasure of epigenetic imprints in iPSCs depending on the nature of chromatin modifications of the original cell type, or in other words, a kind of cell type-specific epigenetic memory [48,49]. However, viral integrations are probably the cause of the partially inconsistent gene expression patterns observed in the LARGE heatmap (Figure 7). In order to identify actual effects of viral integrations on the host cell genome and to avoid genomic alterations, the generation and comparative characterization of non- viral iPSCs, particularly from human AFCs, still remains. In addition to the above-mentioned results of our LARGE analysis, we identified ESC-specific genes in the Venn diagrams, including, for example, PRDM14, WNT3A and GSC. PRDM14 has been implicated in maintaining the undifferentiated ESC state [73]. In contrast, WNT3A and GSC are primitive streak/ mesendoderm markers known to regulate developmental processes [43]. These genes distinguish our AFiPSCs and FiPSCs from ESCs, thus implying incomplete reprogramming and emphasizing general differences between ESCs and iPSCs despite the acquisition of the ESC phenotype in both iPSC types. Follow-up studies should be designed to identify functional consequences of this observation. Conclusion Both, primary AFCs, in particular stem cell-like subpopulations of primary AFCs, as well as AFiPSCs are considered to be valuable for the application in basic and applied research. Taken together, our results propose that, for these purposes, cellular reprogram- ming of AFCs is beneficial as it represses senescence and leads to a phenotype very similar, though not identical, to ESCs. These findings are even more significant, considering that due to the presence of fetal stem cells within bulk primary AFCs, amniotic fluid seems to be a very suitable source of cells for the realization of non-integrating reprogramming strategies. Yet, as a main result of this study, we identified gene expression signatures and LARGE patterns for different types of iPSCs, corresponding parental cells and ESCs. Two conclusions can be drawn from this. First, this kind of comparative transcriptome analysis should be extended integrating iPSC lines derived from several distinct cell sources and generated using various reprogramming techniques, as it would aid in enhancing our meagre understanding of mechanisms underlying cellular reprogramming. Secondly, the functional relevance of such distinct expression patterns, especially of AFCs, AFiPSCs and ESCs, will have to be investigated profoundly in order to estimate limitations and to exploit the full potential associated with putative future utilization of amniotic fluid-derived cells. Supporting Information Table S1 List of primers used for qRT-PCR and DNA fingerprinting analyses. Found at: doi:10.1371/journal.pone.0013703.s001 (0.03 MB XLS) Table S2 List of antibodies used for immunofluorescence stainings. Found at: doi:10.1371/journal.pone.0013703.s002 (0.02 MB XLS) References The LARGE Principle in AFiPSCs Table S3 Complete gene lists reported in the ESC/AFiPSC/ AFC-Venn diagram (Figure 4C). Spreadsheet 1 a–c: Self-renewal signature, 1299 genes, (a) gene list (GL), (b) DAVID functional annotation chart (DFACha), (c) DAVID functional annotation clustering (DFAClu). Spreadsheet 2 a–c: ESC-specific genes, 257 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 3 a–c: AFiPSC-specific genes, 555 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 4 a–b: Housekeeping genes, 6934 genes, (a) GL, (b) DFACha. Spreadsheet 5 a–c: Overlap ESCs and AFCs, 125 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 6 a–c: Donor cell memory, 350 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 7 a–c: Donor cell-specific genes, 665 genes, (a) GL, (b) DFACha, (c) DFAClu. Found at: doi:10.1371/journal.pone.0013703.s003 (5.87 MB XLS) Table S4 Senescence-associated genes. List of 116 senescence- associated genes derived from the Gene Ontology database [35], including those described by Vaziri et al. [45]. These genes served as input for the differential gene expression analysis between AFCs (P17) and the group of all AFiPSC lines. Found at: doi:10.1371/journal.pone.0013703.s004 (0.04 MB XLS) Table S5 Complete gene lists reported in the ESC/FiPSC/Fibs- Venn diagram (Figure 6A). Spreadsheet 1 a–c: Self-renewal signature, 922 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 2 a–c: ESC-specific genes, 645 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 3 a–c: FiPSC-specific genes, 572 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 4 a–b: Housekeeping genes, 6766 genes, (a) GL, (b) DFACha. Spread- sheet 5 a–c: Overlap ESCs and Fibs, 282 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 6 a–c: Donor cell memory, 728 genes, (a) GL, (b) DFACha, (c) DFAClu. Spreadsheet 7 a–c: Donor cell-specific genes, 668 genes, (a) GL, (b) DFACha, (c) DFAClu. Found at: doi:10.1371/journal.pone.0013703.s005 (6.57 MB XLS) Table S6 Complete list of 525 core self-renewal genes as reported in Figure 6B. Spreadsheet a–c: Core self-renewal signature, 525 genes, (a) GL, (b) DFACha, (c) DFAClu. Found at: doi:10.1371/journal.pone.0013703.s006 (0.67 MB XLS) Acknowledgments The authors would like to thank the other members of the Adjaye lab for support and fruitful discussion and A. Sabah at the microarray facility. We would also like to thank Prof. Dr. Wegner and PD Dr. Stumm of the prenatal diagnosis clinic (Zentrum fu¨r Pra¨nataldiagnostik, Kudamm-199, Berlin, Germany) for providing AFCs. We further acknowledge Dr. Korinna Jo¨hrens (Institute for Pathology, Charite´ - Universita¨tsmedizin Berlin, Berlin, Germany) for the interpretation of histological structures. Author Contributions Conceived and designed the experiments: KW JA. Performed the experiments: KW YW. Analyzed the data: KW JA. Contributed reagents/materials/analysis tools: YW AP KS HL JA. Wrote the paper: KW JA. 1. Serr DM, Sachs L, Danon M (1955) The diagnosis of sex before birth using cells from the amniotic fluid (a preliminary report). Bull Res Counc Isr 5B: 137– 138. 2. Mosquera A, Fernandez JL, Campos A, Goyanes VJ, Ramiro-Diaz J, et al. (1999) Simultaneous decrease of telomere length and telomerase activity with ageing of human amniotic fluid cells. J Med Genet 36: 494–496. PLoS ONE | www.plosone.org 13 October 2010 | Volume 5 | Issue 10 | e13703