Adjaye1,4* Abstract The LARGE Principle of Cellular Reprogramming: Lost, Acquired and Retained Gene Expression in Foreskin and Amniotic Fluid-Derived Human iPS Cells Katharina Wolfrum1,2, Ying Wang 1 , Alessandro Prigione1, Karl Sperling 3 , Hans Lehrach1, James 1 Molecular Embryology and Aging Group, Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics, Berlin, Germany, 2 Institute of Chemistry and Biochemistry, Department of Biology, Chemistry, and Pharmacy, Freie Universita¨t Berlin, Berlin, Germany, 3 Institute of Human Genetics, Charite´ - Universita¨tsmedizin Berlin, Berlin, Germany, 4 Stem Cell Unit, Department of Anatomy, Medical College, King Saud University, Riyadh, Saudi Arabia Human amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnostics procedures. Recently, it has been illustrated that these cells may also serve as a valuable model system to study developmental processes and for application in regenerative therapies. Cellular reprogramming is a means of assigning greater value to primary AFCs by inducing self- renewal and pluripotency and, thus, bypassing senescence. Here, we report the generation and characterization of human amniotic fluid-derived induced pluripotent stem cells (AFiPSCs) and demonstrate their ability to differentiate into the trophoblast lineage after stimulation with BMP2/BMP4. We further carried out comparative transcriptome analyses of primary human AFCs, AFiPSCs, fibroblast-derived iPSCs (FiPSCs) and embryonic stem cells (ESCs). This revealed that the expression of key senescence-associated genes are down-regulated upon the induction of pluripotency in primary AFCs (AFiPSCs). By defining distinct and overlapping gene expression patterns and deriving the LARGE (Lost, Acquired and Retained Gene Expression) Principle of Cellular Reprogramming, we could further highlight that AFiPSCs, FiPSCs and ESCs share a core self-renewal gene regulatory network driven by OCT4, SOX2 and NANOG. Nevertheless, these cell types are marked by distinct gene expression signatures. For example, expression of the transcription factors, SIX6, EGR2, PKNOX2, HOXD4, HOXD10, DLX5 and RAXL1, known to regulate developmental processes, are retained in AFiPSCs and FiPSCs. Surprisingly, expression of the self-renewal-associated gene PRDM14 or the developmental processes-regulating genes WNT3A and GSC are restricted to ESCs. Implications of this, with respect to the stability of the undifferentiated state and long-term differentiation potential of iPSCs, warrant further studies. Citation: Wolfrum K, Wang Y, Prigione A, Sperling K, Lehrach H, et al. (2010) The LARGE Principle of Cellular Reprogramming: Lost, Acquired and Retained Gene Expression in Foreskin and Amniotic Fluid-Derived Human iPS Cells. PLoS ONE 5(10): e13703. doi:10.1371/journal.pone.0013703 Editor: Zhongjun Zhou, The University of Hong Kong, Hong Kong Received July 13, 2010; Accepted October 6, 2010; Published October 29, 2010 Copyright: 2010 Wolfrum et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors acknowledge support from the Bundesministerium f u¨r Bildung und Forschung (BMBF, Federal Ministry of Education and Research, www. bmbf.de; A.P.: 01GN0807; Y.W., J.A.: 0315398G) and the Max Planck Society (www.mpg.de). Contributions of K.W. were also made possible by funding from the Deutsche Forschungsgemeinschaft (DFG, www.dfg.de) through the Berlin-Brandenburg School for Regenerative Therapies (BSRT, www.bsrt.de) GSC 203. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: adjaye@molgen.mpg.de Introduction Human amniotic fluid cells (AFCs) represent a heterogeneous mixture of cells originating from different fetal tissues. They have been used for prenatal diagnosis of various congenital fetal abnormalities for more than fifty years [1]. Yet, especially within the last decade, molecular biology-based studies have revealed remarkable features of distinct subpopulations within bulk AFCs. For instance, in 1999, activity of the telomere-elongating enzyme telomerase was detected in young AFCs, with decreasing activity in aged AFCs [2]. Later on, the presence of cells exhibiting certain embryonic stem cell (ESC) features among bulk primary AFCs was reported [3,4]. Other groups have demonstrated the existence of mesenchymal stem cells (MSCs) within the amniotic fluid [5]. Based on these observations, several strategies have been developed to sort stem cell-like populations out of bulk AFCs and different subpopulations have been characterized in more detail [6–10]. Multipotent properties [6,7,11,12] and potential immune-privileged characteristics of particular AFCs [13,14] support the idea of utilizing amniotic fluid as a source of fetal stem cells, with feasible application in regenerative medicine, especially in fetal tissue engineering approaches [13]. However, there are drawbacks associated with the use of AFCs for such purposes. For instance, primary cultures of bulk AFCs, like primary cell lines in general, senesce after prolonged culture periods in vitro. Besides, the fact that AFCs do not form teratomas in vivo [6,15,16] implies that not even the stem cell-like cells within the amniotic fluid are bona fide pluripotent cells. Hence, their ability to form complex, mature differentiated cell types may be restricted. Indeed, the capacity of AFCs to form specialized cell types and to contribute to the formation of certain tissues or organs in vitro and in xenotransplantation experiments in vivo is a subject of debate [6,11,17–23]. We believe that a means of assigning amniotic fluid cells greater value as an in vitro model system to investigate developmental processes, to conduct disease modeling, toxicological studies, drug research and exploitation in regenera- PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13703