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    • Many protocols include the inhibition

    2018-11-08

    Many protocols include the inhibition of BMP, Nodal and Wnt signaling pathways to achieve neural induction (Fig. 2) (for review see Gaspard and Vanderhaeghen, 2010). To this end, signaling inhibitors such as Noggin, Lefty (Lefty1, Lefty2, or LeftyA) and Dickkopf-1 (Dkk-1) have all been used to promote ESC differentiation to early neurectodermal derivatives (Eiraku et al., 2008; Pera et al., 2004; Perrier et al., 2004; Smith et al., 2008; Verani et al., 2007; Watanabe et al., 2005; Watanabe et al., 2007). These inhibitors block mesendoderm formation and permit the adoption of a default neurectodermal differentiation program. In mESCs, Kamiya and colleagues have shown that the zinc-finger nuclear protein, Zfp521, is an important BMP molecular regulator during early neurectodermal specification (Kamiya et al., 2011). In addition, recent work suggests that fibroblast growth factor-2 (FGF2) can also enhance neural induction, acting at a stage of differentiation akin to primitive ectoderm (epiblast) (Cohen et al., 2010). However, it is also important to bear in mind the species differences that may exist in the emergence of neuroepithelial/neuroectoderm (NE) precursors. For example, recent studies by Zhang and colleagues revealed a novel role for PAX6 in early human, but not mouse NE specification (Zhang et al., 2010). Their study demonstrated that PAX6 was a transcriptional determinant of human NE and that distinct isoforms coordinated the transition from pluripotency to the NE fate by inhibiting pluripotent and activating NE expressed genes respectively (Zhang et al., 2010). After neural induction, a second series of growth factors has been used to direct differentiation towards neural subtypes (Fig. 2). For example, early studies suggested that continued exposure to FGF favored neuronal differentiation, while formation of glial actin inhibitor was promoted by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) (Barberi et al., 2003; Reubinoff et al., 2001). Reubinoff and colleagues also reported that the extra-cellular matrix components, laminin and fibronectin, influenced the balance between neuronal or glial differentiation (Reubinoff et al., 2001). Furthermore, several factors have been shown to direct ESC-derived neural precursors towards region specific cell types, including retinoic acid (RA) (caudal, posterior), sonic hedgehog (ventral) and Wnts (dorsal) (reviewed in Erceg et al., 2009). Despite the success of protocols in initiating neural induction from ESCs, the large-scale application of these methodologies is constrained by the high cost of recombinant growth factors. Because of this, recent studies have explored the use of small molecules to induce step-wise transitions from undifferentiated ESCs toward neural cell types (Ding and Schultz, 2004; Ding et al., 2003). In this context, Chambers and colleagues demonstrated that hESCs could be efficiently converted into neural derivatives by employing a combination of the BMP inhibitor Noggin and the small molecule Activin signaling antagonist, SB431542 (Chambers et al., 2009). In this system, Noggin and SB431542 collaborate to block the SMAD signaling required for mesendoderm formation, thus promoting neural differentiation (Chambers et al., 2009).
    Generation of cortical interneurons from ESCs Within the developing forebrain, cells are identified based on their regional, morphological, and positional characteristics in combination with their gene expression patterns. These attributes vary during the course of development, indicating that components contributing to the identification of a specific cell are also time dependent. However, the critical inputs of embryonic positional information are not available in an in vitro culture. Therefore, assignment of cellular identity relies more heavily on the gene expression profile of the cell in question, as well as aspects of its developmental history. In the case of hESC-derived cell types, detailed knowledge of the changes in gene expression during the genesis of human neural lineages is extremely limited. As a consequence, classification of such cell types is based upon the gene expression profiles of ‘corresponding’ cells within the developing mouse brain. This approach, however, does not account for potential species differences in the biology of the human and mouse forebrain development. With these caveats in mind, Fig. 3 summarizes key genes whose expression may be utilized to classify forebrain cells generated from differentiating ESCs.