the large principle of cellular reprogramming

The LARGE Principle of Cellular Reprogramming: Lost, Acquired and Retained Gene Expression in Foreskin and Amniotic Fluid-Derived Human iPS Cells Katharina Wolfrum 1,2 , Ying Wang 1 , Alessandro Prigione 1 , Karl Sperling 3 , Hans Lehrach 1 , James Adjaye 1,4 * 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 Abstract Human amniotic flui d cells (AFCs) are rout inel y obtained for prenatal diagnostics procedures. Recentl y, 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 pluripotenc y and, thus, bypassi ng senesc ence. 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 compa rative transcri ptome 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, know n to regu lat e dev elopmenta l proces ses , are ret ained in AFiPSCs and FiPS Cs. 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-te rm differe ntiati on potenti al 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: ß 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 fu ¨ 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: [email protected] Introduction Human amniotic fluid cells (AFCs) represent a heterogeneous mixture of cells originating from different fetal tissues. They have bee n used for pre natal dia gnosis of var ious congenita l fet al abnormalities for more than fifty years [1]. Yet, especially within the last decade, molec ular biology-bas ed studie s 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 mes enc hymal stem cel ls (MSCs) wit hin the amniot ic fluid [5] . Base d on thes e observati ons, se ver al st rategi es have been deve lope d to sor t ste m cel l-l ike populat ion s out of bulk AFCs and dif fer ent subp opul ations hav e bee n cha racteri zed in mor e detail [6–10]. Multip otent propert ies [6,7, 11,12 ] and potenti al immune -pr ivi leg ed cha rac ter ist ics of par tic ula r AFCs [13 ,14] support the idea of utiliz ing amnio tic fluid as a source of fet al stem cel ls, wit h fea sible appl ica tion in reg ene rati ve medici ne, especi ally in fetal tissue engine ering approache s [13]. However, there are drawba cks associ ate d wit h the use of AFCs for such purposes. For ins tance, pri mar y cul ture s of bul k AFCs, lik e pri mar y cel l lin es in genera l, senesc e aft er prolonged cul ture 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 compl ex , ma tur e di ff erenti at ed 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 debat e [6,11,17–23]. We bel ieve that a means of assi gning 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

the large principle of cellular reprogramming

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