br Results br Discussion In this study we

Results
Discussion In this study we present evidence and show that hPSCs mostly adopt a rostral-dorsal identity during in vitro neural differentiation with both EB and AD paradigms in the absence of patterning morphogens. SHH patterns in vitro differentiated human neuroectoderm under both EB and AD conditions to ventral progenitors, as demonstrated by a PAX6 to NKX2.1 expression shift. However, differential ventral fates are acquired in hPSCs under EB versus AD conditions in response to SHH stimulation (Figure 7H). An MGE regional fate followed by GABA and cholinergic neurons will be generated in SHH-treated EB cells, while ventralized AD cells mostly yield FP progenitors and TH+ neurons. Wnts/β-catenin, STAT3, and p38 pathways are crucial for proper FP specification, and blocking these pathways could largely switch FP to MGE (Figure 7H). Fetal mesencephalic or striatal tissues have been used as a source of DA neurons or GABA neurons for transplantation in clinical trials (Lindvall et al., 1989; Philpott et al., 1997). These clinical studies raise the hope of curing hitherto intractable human neurodegenerative diseases through replacement therapy. Recent advances in targeted differentiation of hPSCs to neuronal subtypes have begun to confirm their therapeutic potential in epilepsy, Huntington disease, Parkinson\'s disease, and Alzheimer\'s disease (Cunningham et al., 2014; Liu et al., 2013; Ma et al., 2012; Roy et al., 2006; Yue et al., 2015). Obtaining functional neuronal subtypes is the major roadblock in regenerative medicine for treating these neurological disorders. Here, we provide a framework for the generation of distinct ventral neuronal subtypes of either FP or MGE origin through a combination of differentiation paradigms and small molecules. These human MGE and FP progenitors as well as their differentiated progeny will thus serve as invaluable cellular sources for the study of adenosine receptor agonist development and related diseases.
Experimental Procedures
Author Contributions
Introduction Fibroblast-to-neuron reprogramming has been demonstrated using transcription factors (Vierbuchen et al., 2010; Pang et al., 2011) and signaling molecules (Hu et al., 2015; Li et al., 2015). These factors jump-start transcriptional programs that redefine cellular identity; however, the potency of each reprogramming factor is cell age and lineage dependent (Liu et al., 2013; Masserdotti et al., 2015; Mertens et al., 2015). Therefore, mechanistically, the success of neuronal reprogramming depends upon the cell-of-origin genetic and epigenetic environment, as well as the ability of reprogramming factors to navigate that environment and induce reprogramming. For instance, the pioneer activity of ASCL1 and the permissible chromatin structures of mouse embryonic fibroblasts facilitate rapid neuronal reprogramming (Wapinski et al., 2013). Likewise, direct competition for pro-neural genetic elements between endogenous repressor complexes and overexpressed neurogenin 2 (NEUROG2) regulates reprogramming of mouse astroglia in short versus prolonged culture (Masserdotti et al., 2015). NEUROG2 is a basic helix-loop-helix transcription factor that promotes early neurogenesis (Ma et al., 1996; Fode et al., 1998; Scardigli et al., 2001). NEUROG2 overexpression in mouse embryonic stem cells and cultured mouse cortical astroglia catalyzes conversion into functional, synapse-forming glutamatergic neurons (Berninger et al., 2007; Heinrich et al., 2010; Thoma et al., 2012). While NEUROG2 is sufficient to induce reprogramming in neural lineage cells, this factor is unable to independently reprogram somatic fibroblasts (Liu et al., 2013; Chanda et al., 2014). The high-efficiency reprogramming of human fetal fibroblasts (MRC-5) into functional cholinergic neurons by NEUROG2 requires simultaneous exposure to the small molecules forskolin, a cyclic AMP (cAMP) synthesis activator, and dorsomorphin, an inhibitor of AMP-activated protein kinase and bone morphogenetic protein type 1 receptors (Liu et al., 2013). Furthermore, the inclusion of SOX11 is required to rapidly convert adult human skin fibroblasts into neurons (Liu et al., 2013). These findings demonstrate that age- and lineage-specific genetic and epigenetic factors directly affect the ability of NEUROG2 to catalyze reprogramming.