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    • Although the exact mechanism by which

    2018-11-08

    Although the exact mechanism by which WNT3 levels in hESCs predict their differentiation potential is not known, we propose a working model that can explain all of our data so far (Figure 3C). It has been established that the identity of hESCs is mainly determined by the core pluripotency regulatory networks (Boyer et al., 2005). Variable levels of lineage factors, such as WNT3, can be tolerated in hESCs, which is supported by the fact that knockdown or overexpression of WNT3 in hESCs does not affect pluripotent gene expression (Figure S2). However, it is likely that higher levels of WNT3 could result in upregulation of multiple factors critical for DE differentiation, rendering the hESCs poised for DE differentiation. Upon receiving other differentiation signals that downregulate the pluripotency transcription network, hESCs could initiate differentiation toward DE. Consistent with this hypothesis, a recent study demonstrated that although constitutive overexpression of SOX17 in hESCs does not affect hESC maintenance, it can restrict hESCs to DE lineage differentiation (Séguin et al., 2008). In summary, we have identified WNT3 as a biomarker capable of predicting the DE differentiation potential of hESC lines. WNT3 appears to be a functional marker, because the DE differentiation potential can be modulated by altering WNT3 levels in hESCs. Our study establishes a simple method for predicting the DE differentiation potential of hESCs, which should facilitate efforts to understand and generate stearoyl-coa desaturase cell lineages.
    Experimental Procedures
    Acknowledgments
    Introduction WNT signaling is involved in multiple processes during early development, including the maintenance and/or proliferation of stem and progenitor populations, cell fate specification, segmentation, and dorsal-ventral patterning (Logan and Nusse, 2004). During gastrulation in the mouse, WNT signaling plays a critical role in the generation of mesoderm, with Wnt3-null embryos failing to form a primitive streak (Liu et al., 1999), the structure from which hematopoietic progenitors and all other mesodermal and endodermal lineages emerge (Kinder et al., 1999). The role of WNT signaling has also been examined at later stages of hematopoietic development. Mouse knockout studies indicate that WNT3A is required for the maintenance of long-term hematopoietic stem cell (HSC) and multipotent progenitors and that WNT3A is the critical ligand that activates canonical WNT signaling in fetal liver HSCs (Luis et al., 2010). These findings are in agreement with earlier work suggesting WNT3A can preserve the immature phenotype of HSCs in vitro or can induce stem cell characteristics in hematopoietic progenitors (Malhotra et al., 2008). Indeed, recent studies utilizing mice carrying hypomorphic alleles of the Apc gene, which binds the WNT signaling intermediate, β-catenin, showed that WNT levels regulate HSCs as well as myeloid and T lymphoid progenitors (Luis et al., 2011). These investigators determined that increasing levels of WNT signaling enhanced T cell differentiation and eventually depleted HSCs due to reduced self-renewal (Luis et al., 2011). Although difficult to study in vivo, the critical early stages of hematopoietic lineage commitment and development can be modeled in vitro using embryonic stem cell (ESC) differentiation. Studies have confirmed that WNT signaling is required for mesoderm formation from differentiating ESCs and for the subsequent emergence of hematopoietic progenitors from mouse (Cheng et al., 2008; Gadue et al., 2006; Jackson et al., 2010; Lako et al., 2001; Lengerke et al., 2008; Lindsley et al., 2006; Nakanishi et al., 2009; Nostro et al., 2008) and human (Murry and Keller, 2008; Sumi et al., 2008; Vijayaragavan et al., 2009; Wang and Nakayama, 2009; Woll et al., 2008) ESCs. The literature cited above underscores the requirement for WNT signaling at different points during the genesis of the hematopoietic system. However, many prior differentiation studies included either stromal layers or undefined media components (Cheng et al., 2008; Gadue et al., 2006; Lako et al., 2001; Lindsley et al., 2006; Vijayaragavan et al., 2009; Woll et al., 2008), raising the possibility that some of the observed effects of WNTs resulted from complex interactions with unknown factors. To address this issue, we developed a defined medium (APEL) that allows the activity of exogenously added factors to be assessed free from the influence of uncharacterized media components, including bovine serum albumin (BSA), knockout serum replacer (KOSR), or serum (Ng et al., 2008).