for a period of five days or a combination of 10 ng/ml BMP4 and 10 mM SB431542 (a TGFbRI inhibitor, Sigma-Aldrich) for 7 days. Undifferentiated controls were grown in N2B27 including 20 ng/ml bFGF only. After a period of 5 d or 7 d, including daily replacement of media, the cells were harvested for RNA isolation for qRT-PCR and global gene expression profiling analyses or fixed for immunofluorescence microscopy analysis. DNA fingerprinting and karyotyping The origin of AFiPSC cell lines 4, 5, 6, 10, and 41 was confirmed by fingerprinting analysis, as previously described [34]. The primer pairs D17S1290 and D21S2055 were used; sequences are provided in Table S1. For the detection of probable karyotypic abnormalities in AFiPSC lines 4, 5, 6, and 41, chromosomal analysis was performed after GTG-banding at the Human Genetic Center of Berlin. For each line, 25 metaphases were counted and 10 karyograms analyzed. Illumina bead chip hybridization and data analyses Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA, www.qiagen.com). In each case, 500 ng RNA were used as input for the bead chip hybridization (Illumina, San Diego, CA, USA, www.illumina.com). Processing of samples and the conversion of raw data was previously described [31]. For correlation coefficient analysis and the generation of Venn diagrams, detected gene expression was defined by a Detection P-Value ,0.01. To be considered as differentially expressed, genes had to be at least 1.5 fold up- or down-regulated in a group-wise comparison of all AFiPSC or FiPSC lines with either AFCs at passage 6 or 17 or fibroblasts (Fibs), respectively. Accordingly, the FDR-adjusted P-Value for differential gene expression had to be ,0.05. Human senescence-associated genes were derived using the AmiGO Browser version 1.7 of the Gene Ontology database (http://www.geneontology.org, 28th of March 2010) [35]. Func- tional annotation and enrichment analyses were done using the DAVID platform version 6.7 (http://david.abcc.ncifcrf.gov/ home.jsp) [36,37]. Illumina ProbeIDs were used as input against the background of the Homo Sapiens species; analyses were executed based on DAVID default parameter settings (19th of April 2010). Quantitative real-time polymerase chain reaction and data analyses Quantitative real-time PCR (qRT-PCR) was performed and analyzed using ABI PRISM SDS 2.1 software (Applied Biosys- tems, Foster City, CA, USA, www.applied biosystems.com) and Microsoft Excel as described elsewhere [31]. All primer sequences are provided in Table S1. For detection of pluripotency-associated genes in AFiPSCs, the undifferentiated ESC line H1 was used as a positive control. For the analysis of BMP2- or BMP4-induced trophoblast differentiation of AFiPSCs, placental RNA (Clontech, Mountain View, CA, USA, www.clontech.com) was used as a positive control. Data are presented as mean LOG2 ratios with respect to biological controls and standard deviation. Immunofluorescence, alkaline phosphatase and cellular senescence staining For the identification of ESC markers in undifferentiated AFiPSCs (lines 4, 5, 6, 10 and 41) and detection of lineage markers in AFiPSCs differentiated in vitro, cells were fixed, permeabilized and stained for immunofluorescent imaging as described by Prigione et al. [31]. The list of primary and secondary antibodies used is provided in Table S2. Nuclei were counter-stained with The LARGE Principle in AFiPSCs DAPI (100 ng/ml, Vector Laboratories, Burlingame, CA, USA, www.vectorlabs.com). Alkaline phosphatase (AP) staining of all manually picked AFiPSC lines was performed following the manufacturer’s instructions (Millipore, Billerica, MA, USA, www.millipore.com). For staining of senescent cells, the Senescence beta-Galactosi- dase Staining Kit (Cell Signaling, Danvers, MA, USA, www. cellsignal.com) was used according to the manufacturer’s protocol. All stainings were visualized and images were acquired using the confocal microscope LSM 510 Meta (Carl Zeiss, Oberkochen, Germany, www.zeiss.de). Processing of images was done with the help of AxioVision V4.6.3.0 (Zeiss) and Adobe Photoshop CS version 8.0 (Adobe, Munich, Germany, www.adobe.com) software. Results Senescence is bypassed by the derivation of AFiPSCs from human AFCs Under routine cell culture conditions, bulk primary human AFCs (Figure 1A-I) senesce at approximately passage 17 as determined by decelerated proliferation. These cells also have an enlarged and flattened morphology and stain positive for the senescence-associated beta-galactosidase (Figure 1A-II, -III). To bypass senescence and to enhance proliferation capacities of AFCs, we derived iPSCs from primary bulk AFCs by transduction with a retroviral cocktail consisting of OCT4, SOX2, KLF4 and c- MYC (OSKM) [24]. The resulting AFiPSC colonies appeared about seven days post-transduction (Figure 1A-IV), which is approximately two weeks earlier than what we and others have observed for fibroblast-derived iPSCs [24,31]. Five clonal AFiPSC lines were expanded under ESC conditions and partly character- ized. Of these, two lines underwent complete characterization. AFiPSCs were indistinguishable from ESCs (e.g. ESC line H1) in terms of morphology (Figure 1A-V) and proliferation. These AFiPSCs also resembled ESCs with respect to alkaline phospha- tase (AP) activity and expression of several markers of the undifferentiated state, including NANOG, OCT4, SOX2, SSEA4, TRA-1-60, TRA-1-81 as determined by immunocytochemistry (Figure 1B). The AFiPSCs exhibited a normal karyotype several passages after their generation (Figure 1C) and their genetic relatedness to primary AFCs cells was confirmed by DNA fingerprinting analysis (Figure 1D). Pluripotency and in vitro and in vivo differentiation of AFiPSCs Microarray-based transcriptional analysis revealed up-regula- tion of self-renewal and pluripotency-associated genes [38–40] in AFiPSCs in contrast to primary AFCs (Figure 2A). qRT-PCR validations, performed for a selection of these pluripotency- associated genes, confirmed the array-derived data (Figure 2B). The ability of AFiPSCs to differentiate into derivatives represen- tative of all three embryonic germ layers was assessed by embryoid body differentiation in vitro (Figure 2C) and teratoma formation in vivo (Figure 2D). Markers or histological structures representing endoderm-, mesoderm- and ectoderm-derived lineages were detected in both assays (Figure 2C, D). Trophoblast differentiation of AFiPSCs To test if AFiPSCs, like ESCs, can undergo trophoblast differentiation [41–44], we stimulated two AFiPSC lines with 100 ng/ml BMP2 or BMP4 over a period of five days. As a result, a morphological change from densely packed ESC-like colonies (Figure 3A-I, -II, -V, -VI) towards more loosely packed clusters of PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13703