In summary we have dissected the specific

In summary, we have dissected the specific role that OCT4 plays during the first steps of reprogramming independently of the other reprogramming factors. We have shown that OCT4 does not require SOX2 to interfere with cell-type-specific gene-expression profiles or to initiate an unstable transcriptional state that facilitates reprogramming. Finally, we have discovered a mechanism by which OCT4 and KLF4 compete to differentially regulate Mgarp expression, resulting in distinct expression levels at different stages of reprogramming. Therefore, our study sheds new light on the function of OCT4 in the reprogramming process.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction Today’s human induced pluripotent stem cell (hiPSC)-based disease and toxicology screening efforts and tomorrow’s hiPSC-based auto/allogeneic cell therapies will require robust, reproducible methods of cell line generation and expansion, without the integration of the reprogramming factor transgenes (Saha and Jaenisch, 2009). hiPSCs were first generated by the ectopic expression of multiple genes introduced through genome-integrating retro- and lentiviral expression systems: namely OCT4 (POU5F1), SOX2, cMYC, KLF4, LIN28, and NANOG (Takahashi et al., 2007; Yu et al., 2007). To eliminate as many integrating events as possible, several studies were able to substitute unique small molecule inhibitors for a number of reprogramming factors (Nie et al., 2012). The derivation of footprint-free hiPSCs has been demonstrated using several nonintegrative strategies, including episomal-, RNA-, Sendai virus-, and protein-based methods (Fusaki et al., 2009; Warren et al., 2010; Yu et al., 2009; Zhou et al., 2009). However, nonintegrative methods have proven to be inefficient and labor intensive, often requiring additional reprogramming factors (Lee et al., 2013). In the most commonly used conventional culture system, human embryonic stem clozapine-n-oxide (hESCs) and hiPSCs are maintained on feeder cells while passaged as clumps to prevent extensive cell death and genomic aberrations (Thomson et al., 1998). The inability to single-cell culture hiPSCs in a feeder-free (FF) environment severely limits potential industrial scale screening or cell therapy applications (Valamehr et al., 2011). Recent efforts focused on improving single-cell survival have identified small molecule inhibition of the Rho-associated protein kinase to dramatically reduce cell death upon single-cell dissociation (Watanabe et al., 2007; Xu et al., 2010). More recently, we have demonstrated efficient single-cell and FF culture of lentiviral-derived hiPSCs in the presence of a small molecule cocktail, SMC4 (Valamehr et al., 2012). By inhibiting pathways associated with cell death and differentiation, we were able to achieve high-resolution direct flow cytometry sorting and maintenance of hiPSCs in a completely FF system (Abujarour et al., 2013; Valamehr et al., 2012). However, our study focused on lentiviral-derived hiPSCs that were not transgene-free, limiting its therapeutic relevance. Another challenge in pluripotent stem cell culture is their propensity for spontaneous differentiation (Pera and Trounson, 2004; Valamehr et al., 2011). This issue has been resolved for mouse pluripotent stem cells: by blocking differentiation cues of mouse ESCs in culture through small molecule inhibition of mitogen-activated protein kinase and glycogen synthase kinase 3 (termed “2i”), the ground state of pluripotency was achieved, and spontaneous differentiation of mouse ESCs was prevented (Ying et al., 2008). Similar studies in hESCs have been described; however, continuous ectopic expression of pluripotency genes was necessary to maintain the ground state resulting in genome-modified human pluripotent stem cells (Hanna et al., 2010a; Wang et al., 2011). Here, we describe a multistage culture platform that enables highly efficient episomal reprogramming with a significant reduction in time and effort for hiPSC generation and using just a minimal number of reprogramming factors (OCT4/SOX2/SV40LT). Furthermore, the system can be multiplexed to allow for parallel reprogramming and characterization of multiple somatic cell lines with ease and in an expedited time: bona fide hiPSCs are individually flow cytometry sorted into 96-well plates, characterized, and readily expanded with minimal hands-on effort. We describe improvements in medium composition that allow long-term maintenance of transgene-free hiPSCs in single-cell and FF passage culture with genomic stability and minimal evidence of spontaneous differentiation. Furthermore, gene expression analysis shows that small molecule inhibition of specific signaling pathways may drive hiPSCs to a common ground state of pluripotency, regardless of genetic background and independent of transgene expression. We believe that this demonstration of stable culture of transgene/footprint-free hiPSCs as single cells in a FF environment with the elimination of spontaneous differentiation will serve as a valuable asset in driving pluripotent stem cell technology toward clinical applications.