AP20187 br Materials and methods br Results br

Materials and methods
Results
Discussion Here we show that continued (albeit prolonged) culture of plastic hFibOCT4 in RM media is sufficient for induction of pluripotency. Importantly, iPSCOCT4 possess morphological, biomolecular, and functional hallmarks of bona fide PSCs (K.R. Salci et al., 2015). Building on our previous works using OCT4 to induce a state of plasticity required for cell fate conversion (R. Mitchell et al., 2014; R. R. Mitchell et al., 2014; Szabo et al., 2010), our current findings highlight that pluripotency represents another cell fate choice for plastic human fibroblasts (Fig. 3) and provides further example that cell fate alteration through plasticity induction can be achieved without additional TFs (Efe et al., 2011; Kim et al., 2011; Li et al., 2013) or small molecules (Wang et al., 2014; Zhu et al., 2014). Despite the myriad of methodologies to induce pluripotency in human somatic AP20187 (Anokye-Danso et al., 2011; Buganim et al., 2014; Eminli et al., 2008; Giorgetti et al., 2009; Heng et al., 2010; Hou et al., 2013; Kim et al., 2009; Li et al., 2011; Montserrat et al., 2013; Nakagawa et al., 2008; Redmer et al., 2011; Shu et al., 2013; Takahashi et al., 2007; Theunissen and Jaenisch, 2014; Yu et al., 2007; Zhu et al., 2010), exogenous delivery or endogenous activation of OCT4 is indispensible for reprogramming to pluripotency. Furthermore, we are unaware of any report suggesting that iPSCs can be generated without the use of pluripotent-supportive reprogramming media. As we have demonstrated that pluripotency is another destination of plastic human fibroblasts, we suggest that conventional reprogramming to pluripotency (Takahashi et al., 2007; Yu et al., 2007) relies on the combination of OCT4-induced plasticity and pluripotent media instruction, and is expedited or further specified by the addition of pluripotent-specifying TFs (SKM, SNL). We also propose that this could explain why pluripotency is transiently acquired during OSKM-mediated “transdifferentiation” (Maza et al., 2014). Given the reduced complexity of our reprogramming system, we feel OCT4 -induced plasticity represents an ideal model to elucidate the governing mechanisms that allow for alteration of cell fate upon manipulation of native transcriptional programs toward both lineage specific progenitors and pluripotent stem cells alike.
Acknowledgments We would like to thank Dr. Borko Tanasevijic and Rami Mechael for their molecular expertise. K.R.S. is funded by NSERC (CREATE M3 Scholarship). R.R.M. is funded by CIHR (GSD 121742). M.B. is supported by the Canadian Research Chair Program in Human Stem Cell Biology, and funded by a CIHR operating grant (MOP-123304) and Marta and Owen Boris Foundation.
Introduction The olfactory neurons renew itself throughout life and have potential application in central nervous system (CNS) repair (Barnett and Riddell, 2004; Li et al., 1997; Roisen et al., 2001). Olfactory mucosa is easily accessible, and can be obtained by a simple biopsy performed through the external nares (Feron et al., 1998). Olfactory stem cells are generated from olfactory mucosa (Murdoch and Roskams, 2008; Murrell et al., 2005; Tome et al., 2009). Various culture conditions generate olfactory stem cells that differ according to species and developmental stage and have different progenitor or stem cell characteristics (Lindsay et al., 2013; Murdoch and Roskams, 2008; Murrell et al., 2005; Tome et al., 2009; Zhang et al., 2004). Murrell et al. reported that cells from human olfactory mucosa generate neurospheres that are multipotent in vitro and human olfactory neurospheres gave rise to neurons and glia and self replicating (Murrell et al., 2005, 2008). Olfactory spheres (OSs) are clusters of progenitors or stem cells generated from olfactory mucosa in suspension culture. We previously reported that adult rat olfactory sphere cells (OSCs) were generated, expressed oligodendrocyte progenitor cell (OPC) markers, and differentiated into oligodendrocytes in vivo and in vitro (Ohnishi et al., 2013). Under standard culture conditions, adult rat OSCs rarely differentiate into neurons. In the present study, human adult OSCs were generated and their characteristics investigated. Human adult OSCs also expressed OPC markers, but they were directed toward neuronal differentiation in our culture condition. These findings suggest that human OSCs have potential to be used as a cell source of neural progenitors.