Efficient Feeder-Free Episomal Reprogramming with Small Molecules Junying Yu*, Kevin Fongching Chau, Maxim A. Vodyanik, Jinlan Jiang, Yong Jiang Advanced Development Programs, Cellular Dynamics International, Inc., Madison, Wisconsin, United States of America Abstract Genetic reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) could offer replenishable cell sources for transplantation therapies. To fulfill their promises, human iPSCs will ideally be free of exogenous DNA (footprint- free), and be derived and cultured in chemically defined media free of feeder cells. Currently, methods are available to enable efficient derivation of footprint-free human iPSCs. However, each of these methods has its limitations. We have previously derived footprint-free human iPSCs by employing episomal vectors for transgene delivery, but the process was inefficient and required feeder cells. Here, we have greatly improved the episomal reprogramming efficiency using a cocktail containing MEK inhibitor PD0325901, GSK3b inhibitor CHIR99021, TGF-b/Activin/Nodal receptor inhibitor A-83-01, ROCK inhibitor HA-100 and human leukemia inhibitory factor. Moreover, we have successfully established a feeder-free reprogramming condition using chemically defined medium with bFGF and N2B27 supplements and chemically defined human ESC medium mTeSR1 for the derivation of footprint-free human iPSCs. These improvements enabled the routine derivation of footprint-free human iPSCs from skin fibroblasts, adipose tissue-derived cells and cord blood cells. This technology will likely be valuable for the production of clinical-grade human iPSCs. Citation: Yu J, Chau KF, Vodyanik MA, Jiang J, Jiang Y (2011) Efficient Feeder-Free Episomal Reprogramming with Small Molecules. PLoS ONE 6(3): e17557. doi:10.1371/journal.pone.0017557 Editor: Martin Pera, University of Southern California, United States of America Received December 6, 2010; Accepted January 26, 2011; Published March 1, 2011 Copyright: 2011 Yu 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: This study was supported by Cellular Dynamics International, Inc. (http://www.cellulardynamics.com/). The funder had a role in the decision to publish the manuscript. Competing Interests: All authors are employed by, and have a financial interest in, Cellular Dynamics International, Inc.. Additionally, JY is listed as the inventor on a patent application related to the work presented in this paper. This, however, neither interferes with the authors’ objectivity in performing research presented in the manuscript, nor alters the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: jyu@cellulardynamics.com Introduction Human induced pluripotent stem cells (iPSCs), similar to human embryonic stem cells (ESCs), are capable of unlimited proliferation and have the potential to differentiate into all cell types of the body [1–2]. These cells, thus, have applications in basic biology, disease modeling, drug development, and trans- plantation therapies. By expressing a defined set of reprogram- ming factors, iPSCs have been generated from many cell types of different species [1–8]. Initial methods for iPSC generation employed genome-integrating retroviral or lentiviral vectors [2– 3]. These approaches could produce tumorigenic insertional mutations, and residual or reactivation of transgene expression during iPSC differentiation could affect lineage choice and the functionality of iPSC derivatives [2,9]. To overcome these problems, various methods were developed to derive iPSCs free of exogenous DNA (footprint-free), including repeated treatments with reprogramming factors (plasmids, minicircle DNA, non- integrating adenoviral vectors and proteins), transposons and RNA viral vectors [10–16]. However, these methods suffer one or more of the following limitations: the unacceptable low reprogramming efficiency; the labor-intensive removal of reprogramming factors from iPSCs; the requirement for viral packaging or feeder cells. Thus, there is a need to develop a simple and efficient feeder-free method to enable the routine derivation of footprint-free iPSCs from many human donor samples, and eventually the derivation of clinical-grade human iPSCs. A recent report described the efficient derivation of footprint- free human iPSCs from fibroblasts using synthetic modified mRNA [17]. Compared to viral and DNA-based reprogramming methods, the mRNA-mediated transgene delivery offers a safer approach for the derivation of clinical-grade human iPSCs. The requirement for repeated transfections, however, limits the application of this method to cells types that are easily transfectable such as skin fibroblasts. It remains to be seen whether this method can be readily adapted to cells that are relatively resistant to lipid-mediated transfections, such as cells of hematopoietic lineages. In addition to the mutations arising during reprogramming, somatic mutations present in the donor cells may also significantly affect the safety of human iPSCs. Therefore, the selection of appropriate donor cell types will likely be important for the derivation of clinical-grade human iPSCs. A reprogramming method that is applicable to different cell types will be highly desirable to address this question. Additionally, recent data suggest the retention of donor cell epigenetic memory in early passage iPSCs [18], which influences their in vitro differential capacity. It remains to be seen whether this is affected by the specific methods employed in the derivation of iPSCs. Thus alternative methods are needed for the efficient derivation of human footprint-free iPSCs. We have previously generated footprint-free human iPSCs using oriP/EBNA-1 (Epstein-Barr nuclear antigen-1) episomal vectors to deliver reprogramming genes (OCT4, SOX2, NANOG, LIN28, c- MYC, KLF4 and SV40LT) [19]. Compared to other methods, this approach has several advantages. First, the oriP/EBNA-1 vectors PLoS ONE | www.plosone.org 1 March 2011 | Volume 6 | Issue 3 | e17557