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  • br Introduction Foundational cell fate reprogramming

    2018-11-08


    Introduction Foundational cell fate reprogramming studies revealed ectopic expression of pluripotency-associated transcription factors (TF) OCT4, SOX2, KLF4, and c-MYC (OSKM) or OCT4, SOX2, NANOG, LIN28 (OSNL) in human fibroblasts (hFibs) cultured in pluripotent supportive conditions was sufficient for generation of induced pluripotent stem daunorubicin (iPSCs) possessing phenotypic, molecular, and functional characteristics akin to embryonic stem cells (ESCs) (Takahashi et al., 2007; Yu et al., 2007). A multitude of reprogramming cocktails and methodologies have since been demonstrated to yield iPSCs from a range of adult cell types (Theunissen and Jaenisch, 2014). Early modifications revealed ectopic expression of SKM could be functionally replaced by expression of structural homologs (Nakagawa et al., 2008) or chemical compounds (Li et al., 2011; Zhu et al., 2010), and altogether bypassed if they were endogenously expressed in starting cell types (Eminli et al., 2008; Giorgetti et al., 2009; Kim et al., 2009). More recently, groups have identified that Nr5a2 (Heng et al., 2010), E-cadherin (Redmer et al., 2011) and Gata3 (Montserrat et al., 2013; Shu et al., 2013) are capable of substituting for Oct4 when expressed with SKM; while others have replaced OS with Sall4 and Esrrb in combination with NL (Buganim et al., 2014). Furthermore, complete replacement of ectopic expression of OSKM has been achieved using miR302/367 expression in combination with Hdac2 suppression (Anokye-Danso et al., 2011), and a combination of seven small-molecule compounds in the mouse system (Hou et al., 2013). However, despite these advances, a substitute that definitively replaces Oct4\'s functional activity and activates its gene targets in the absence of other reprogramming factors has not been identified. As such, OCT4\'s activity is indispensible for induction and maintenance of pluripotency (Orkin et al., 2008; Sterneckert et al., 2012). Further supporting this concept, ectopic expression of high performance Oct4-VP16 transactivation domain fusion protein in mouse embryonic fibroblasts (MEF) is the only example of single factor reprogramming to achieve germline competent iPSCs without the addition of small molecules, miRNA, or ectopic/endogenous support by additional pluripotent factors (Wang et al., 2011). Using similar cellular reprogramming principles, two paradigms exist toward achieving cell fate conversion. One approach relies on forced expression of lineage-specific TFs to facilitate lineage conversions (Lujan et al., 2012; Vierbuchen et al., 2010). The alternative focuses on inducing an unstable or plastic cell state, demarcated by activation of multiple lineage specific gene expression programs, that is capable of responding to environmental cues (R. Mitchell et al., 2014; Orkin and Hochedlinger, 2011). Strategies using short-term exposure to OSKM (Efe et al., 2011; Kim et al., 2011), OCT4 with small molecule substitutes of SKM (Wang et al., 2014; Zhu et al., 2014), and OCT4 alone (R. Mitchell et al., 2014; R. R. Mitchell et al., 2014; Szabo et al., 2010) in combination with reprogramming media (RM) have been employed toward achieving a cell state that responds to environmental cues. However, these approaches are molecularly distinct as the addition of pluripotency factors SKM convolutes plasticity induction by up-regulating early development and pluripotency genes (Maza et al., 2014; R. Mitchell et al., 2014), indicating that OCT4 alone in combination with short-term exposure to RM is minimally sufficient to induce plasticity (R. Mitchell et al., 2014). Accordingly, OCT4-induced plastic cells are capable of responding to environmental instruction toward derivation of neural and hematopoietic progenitors without traversing pluripotency (R. R. Mitchell et al., 2014; Szabo et al., 2010). Although molecular/functional evidence indicates that transcriptional hallmarks of iPSCs are not observed in OCT4 plastic cells during short-term exposure to RM, whether continued culture in conditions known to support pluripotency is sufficient to up-regulate these programs remains to be elucidated.