br Results br Discussion Here we describe

Results
Discussion Here, we describe a multistage process for highly efficient nonintegrative cellular reprogramming coupled to a method of scalable hiPSC culture that utilizes stage-specific media formulations: FRM for enhanced reprogramming, and FMM for long-term maintenance. Previous studies have demonstrated the versatility of small molecules in the reprogramming and maintenance of pluripotent stem brain metabolism of various species (Nie et al., 2012). However, to date, no small molecule-driven platform has demonstrated the ability to enhance reprogramming and support single-cell and FF culture of footprint-free iPSCs derived from human cells (Nichols and Smith, 2012). We show that the application of specific combinations of small molecule inhibitors in a stage-specific manner is key to enabling robust reprogramming and long-term culture. Studies have indicated that a reprogramming cell population is uniform in the early stages with major variations occurring thereafter (Polo et al., 2012). This suggests that counteracting lineage-specifying pathways early could facilitate the reprogramming process (Polo et al., 2012; Shu et al., 2013). Mesenchymal-to-epithelial transition (MET) has also been shown to support nuclear reprogramming (Li et al., 2010). By blocking differentiation cues early in the reprogramming process and promoting MET through small molecule inhibition of specific pathways, the efficiency of hiPSC generation was significantly improved using the FRM. Indeed, the use of the FRM resulted in robust reprogramming using episomal vectors, in FF and single-cell culture systems, including a demonstration of OCT4/SOX2/SV40LT-only episomal reprogramming. Many ambitious hiPSC banking efforts are currently underway, with the goal of deriving populations of cells from different genetic and disease backgrounds (Rao, 2013). The derivation of footprint-free hiPSCs from various parental somatic sources in a multiplex manner has proven to be difficult and laborious (Narsinh et al., 2011; Saha and Jaenisch, 2009). By significantly improving the efficiency of episomal-induced reprogramming and selecting bona fide hiPSCs by direct flow cytometry sorting into 96-well plate formats at clonal density, cell line generation and characterization can be multiplexed. This system enables a single researcher to parallel reprogram multiple somatic lines using minimal starting cell material and derive many transgene-free hiPSC clones in single-cell, FF culture systems with minimal effort and in an expedited time frame. Long-term passage of pluripotent stem cells in environments applicable to scaled cell production is hampered by spontaneous differentiation and genomic instability (Laurent et al., 2011; Taapken et al., 2011). Preventing spontaneous differentiation while increasing clonality with the use of small molecules has been demonstrated in both mouse and human pluripotent stem cells; however, the latter required ectopic expression of several pluripotency genes resulting in genetic manipulation (Hanna et al., 2010a; Ying et al., 2008). These cells represent the ground state of pluripotency that is associated with a capacity to give rise to all cell types and expression of unique genes associated with core pluripotency (Hanna et al., 2010b; Nichols and Smith, 2012). The more developmentally advanced stage of pluripotency representing the metastable state has been associated with human pluripotent stem cell cultures and is defined by properties that include spontaneous differentiation, high rate of cell death upon single-cell dissociation, and the inability to give rise to all somatic cell types (Nichols and Smith, 2012). We identified inhibition of TGF-β as a contributing factor in the spontaneous differentiation observed in long-term passage of transgene-free hiPSCs. FMM does not include a TGF-β inhibitor and is able to support the continuous maintenance of footprint-free hiPSCs passaged as single cells in a FF environment without the additional requirement of transgene expression. These media and culture systems facilitate culture practices for everyday users, such as removing the requirement for clump passaging, daily culture feeding, or culture cleaning, and therefore deliver a robust basis for a scaled manufacturing process, relevant for future cell therapy applications. We demonstrate that the genomic stability of multiple transgene-free hiPSC lines is supported by FMM after long-term single-cell enzymatic passages in a FF culture environment. This improved viability and stability in single-cell culture will be particularly useful in hiPSC applications such as selection of genomic modifications in disease-correction studies (Saha and Jaenisch, 2009). Collectively, our data suggest that hiPSCs cultured in FMM portray properties commonly associated with the ground state as demonstrated by features such as repression of differentiation gene expression and high clonality; however, additional studies will be necessary to confirm these observations (De Los Angeles et al., 2012; Hanna et al., 2010a, 2010b; Ying et al., 2008). The potential FMM-induced ground state is seen to be independent of transgene expression but also appears to be independent of derivation method, source of parental line, or previously maintained culture conditions. These qualities are lost when hiPSCs are switched from FMM to conventional culture (FMM→Conv) but gained when switched from conventional culture to FMM (Conv→FMM), suggesting that the ground state may be interconvertible as seen previously with mouse pluripotent stem cells (Marks et al., 2012). Two recent advancements in mouse iPSC technology include the elimination reprogramming factors in deriving mouse iPSCs and synchronizing the reprogramming process to nearly perfect efficiencies (Hou et al., 2013; Rais et al., 2013). The need for optimal culture conditions, i.e., 2i, was an important requirement for the success of both of these studies. FMM may represent the human pluripotent stem cell version of 2i and help accelerate the success of current studies. During the preparation of this manuscript, two studies demonstrated the maintenance of pluripotent stem cells in the ground state or resembling preimplantation epiblast using unique small molecule cocktails with similar pathway inhibition as utilized in FMM (Chan et al., 2013; Gafni et al., 2013).