tive medicine could be achieved by cellular reprogramming of these cells to an undifferentiated ground state. As a result, AFCs acquire the ability to self-renew and become pluripotent. The induction of pluripotency was first achieved in human somatic cells employing combinations of retroviral or lentiviral vectors encoding either OCT4, SOX2, KLF4, and c-MYC or OCT4, SOX2, NANOG, and LIN28, respectively [24,25]. Since then, induced pluripotent stem cells (iPSCs) of different somatic origins, from healthy and diseased individuals have been generated using various techniques [26–28]. During the course of this study, the generation of iPSCs from human AFCs were described [15,29,30]. However, these studies failed to characterize amniotic fluid-derived iPSCs beyond the standard assays required to confirm induced pluripotency. Yet, for potential application of iPSCs in basic and applied research, various fundamental aspects of iPSCs, in general, and of this new AF-derived iPSC type, in particular, remain to be understood. Our study aimed at a more detailed molecular characterization of AFiPSCs. To this end, we generated AFiPSCs and demonstrated their ability to differentiate into the extraembryonic trophoblast lineage. This study also highlights the potential of cellular reprogramming to avert replicative senescence observed in bulk primary AFCs. Furthermore, we have analyzed similarities and differences between AFiPSCs, ESC lines H1 and H9 and fibroblast-derived iPSCs (FiPSCs) on the basis of global gene expression. We discuss a fundamental principle of cellular reprogramming, which we have coined LARGE, the Lost, Acquired and Retained Gene Expression principle. This refers to specific genes, which are either switched off, activated or which remain expressed upon induction of pluripotency. In this context, we demonstrate the activation of a common self-renewal and pluripotency-associated gene regulatory network upon cellular reprogramming. Furthermore, we highlight putative implications of the loss of distinct donor cell signature genes and the activation and/or retention of genes implicated in development processes upon cellular reprogramming. Materials and Methods Ethics Statement Auxiliary samples of human AFCs obtained during routine amniocentesis were kindly donated by the clinical laboratory of Prof. Dr. Wegner/PD Dr. Stumm (Zentrum f u¨r Pra¨nataldiagnos- tik, Kudamm-199, Berlin, Germany) after written informed consent. Utilization of these cells was approved by the ethics commission of the Charite´ Universita¨tsmedizin Berlin. Cell culture conditions For the initial culture period (up to passage 5) AFCs were grown in AmnioMAX-C100 (Invitrogen, Carlsbad, CA, USA, www. invitrogen.com). Cells were subsequently cultured in alpha-MEM supplemented with 15% embryonic stem cell-qualified fetal bovine serum, 1% L-glutamine, 1% penicillin/streptomycin (all from Invitrogen), 18% Chang B and 2% Chang C (Trinova Biochem, Giessen, Germany, www.trinova.de) at 37uC in a humidified 5% CO2 atmosphere. AFCs were routinely passaged using 0.05% Trypsin/EDTA (Invitrogen) at varying ratios of 1:3 or even 1:6 when cells were sub-confluent. Human ESC lines H1 and H9 (WiCell Research Institute, Madison, WI, USA, www.wicell.org), AFiPSCs (derived from human AFCs) and FiPSCs (derived from human neonatal foreskin fibroblasts, HFF1 (ATCC, #ATCC-SCRC-1041, Manassas, VA, USA, www.atcc.org)) were cultured under feeder-dependent and feeder-free conditions as described by Prigione et al. [31]. For The LARGE Principle in AFiPSCs comparative transcriptome analysis ESCs and AFiPSCs were adapted to feeder-free culture conditions in mTeSR (Stemcell Technologies, Vancouver, BC, Canada, www.stemcell.com). Retroviral production and iPSC generation OCT4, SOX2, KLF4 and c-MYC retroviruses were generated using pMX vectors as described previously [24]. Briefly, 7.5*10(6) Phoenix Ampho Cells were seeded onto gelatin-coated T75 cell culture flasks and grown in DMEM (Invitrogen) supplemented with 10% FBS (Biochrome, Berlin, Germany, www.biochrom.de) for 16 h. The cells in one flask were then transfected with 12 mg of one of the retroviral DNA vectors encoding either OCT4, SOX2, KLF4 or c-MYC using the FuGENE HD transfection reagent (Roche, Basel, Switzerland, www.roche.ch) according to the manufacturer’s instructions. The retrovirus-containing medium was harvested 48 and 72 h post-transfection. For the generation of AFiPSCs, 180,000 AFCs were transduced with a cocktail of the respective retrovirus-containing media, supplemented with 4 mg/ ml Polybrene (Sigma-Aldrich, Munich, Germany, www.sigmaal- drich.com) at a rate of 1.25 or 2.5 MOI on days 1 and 2 after plating. Each time, directly after the addition of retroviruses, the plates were centrifuged at 8006g, at 37uC for 99 min before replacement of the infectious medium by fresh medium (DMEM/ 10% FBS). The next day, the infected cells were plated onto irradiated MEFs on Matrigel-coated dishes in DMEM/10% FBS. Another 24 h later, the medium was switched to ESC medium [31] for a total period of 10 d, with replacement on alternate days. Afterwards, the infected cells were grown in mouse embryonic fibroblast-conditioned medium (MEF-CM) [32], which was changed at an interval of 2 d until reprogrammed AFiPSC colonies were manually picked 24 d post-transduction and expanded under ESC conditions. Currently, we have AF-derived iPSC lines 4, 5, 6, 10, and 41 in culture passaged more than 25 times (P25). The generation of FiPSCs used for the comparative transcrip- tome analysis has been described [31]. In vitro and in vivo differentiation of AFiPSCs For in vitro differentiation, embryoid body (EB) formation of AFiPSC lines 4, 5 and 41 was induced in ESC medium without bFGF supplementation using the hanging-drop method [33]. After 2 to 3 d, EBs were placed onto low-attachment dishes. A week later, EBs were plated onto gelatin-coated dishes, allowed to differentiate for an additional 10 to 14 d and then stained. In vivo differentiation experiments were performed by EPO-Berlin GmbH (Germany, www.epo-berlin.de). Basically, approximately 2*10(6) cells of the AFiPSC lines 4 and 41 were collected by type IV collagenase-treatment or 0.05% Trypsin/EDTA-treatment, washed, pooled and injected s.c. into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice, commonly known as NOD scid gamma (NSG). Teratomas were collected approximately 63 d after injection and processed according to standard procedures for paraffin embedding and hematoxylin and eosin staining. Histo- logical analysis was performed by a pathologist. Trophoblast differentiation of AFiPSCs To induce differentiation into the trophoblast lineage, AFiPSC lines 5 and 41 were transferred onto Matrigel-coated cell culture dishes and grown in MEF-CM including 8 ng/ml bFGF (PeproTech, Rocky Hill, NJ, USA, www.peprotech.com) until they attained about 30 to 50% confluency. At this point, medium was changed to defined N2B27 medium (Invitrogen) lacking bFGF but including either 100 ng/ml BMP2 (PeproTech) or BMP4 (R&D Systems, Minneapolis, MN, USA, www.rndsystems.com) PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13703