br Results br Discussion Here we performed genome

Results
Discussion Here we performed genome-wide ATAC-seq, ChIP-seq, and RNA-seq profiling to define the immediate-early molecular events that catalyze fibroblast-to-neuron reprogramming by NEUROG2 and small molecules. While NEUROG2 is sufficient to reprogram glial fccp into neurons, this transcription factor fails to reprogram somatic fibroblasts independent of the small molecules F and D (Liu et al., 2013). This synergistic reprogramming activity is the result of extensive SOX4-mediated chromatin remodeling that enhances NEUROG2 occupancy at pro-neural genetic elements and, in coordination with downstream factors, redefines the fibroblast transcriptome. These findings enabled us to reprogram adult fibroblasts and glioblastoma cells refractory to conversion by NEUROG2 alone.
Experimental Procedures
Author Contributions D.K.S., conceptualization, data curation, formal analysis, investigation (Figures 1, 2, 3, 4, 5, 6A–6H, 7, and S1–S4), methodology, software, supervision, validation, visualization, writing – original draft, and writing – review and editing; J.Y., investigation (Figures 6G–6I); M.-L.L., supervision; C.-L.Z., conceptualization, funding acquisition, resources, and supervision.
Acknowledgments We thank Mark Borromeo, Bradford Casey, Jane E. Johnson, Stephen Johnson, Taekyung Kim, Zhigao Wang, and members of the Zhang laboratory. This work was supported by the Decherd Foundation, the Mobility Foundation, the NIH (NS070981, NS092616, NS093502, and NS088095), the Texas Institute for Brain Injury and Repair, and the Welch Foundation (I-1724).
Introduction Human embryonic, induced pluripotent, and adult stem cells are invaluable tools for drug discovery, human toxicology, and studies on human development. Through controlled stem cell differentiation, large quantities of cell types from various tissues have been generated, including lung, liver, intestine, and neural to name a few (Lancaster et al., 2013; Spence et al., 2011; Takebe et al., 2013; Wetsel et al., 2011). In all cases, expansion in a stem or progenitor cell state is required to achieve large cell numbers, followed by differentiation into terminally differentiated fates. Such expansion and differentiation processes depend on myriad soluble factors, and cellular interactions with other cells and the extracellular matrix (ECM). This complexity has motivated the development of high-throughput screening tools to explore the large combinatorial signaling “space” associated with stem cell differentiation. The development of high-throughput screening platforms that emulate the complexity of natural stem cell microenvironments has provided basic insights into stem cell regulation, as well as enabling numerous applications (Lutolf et al., 2009; Soen et al., 2006). For example, toxicity screening systems that use human stem/progenitor cells and their terminally differentiated derivatives may help improve preclinical characterization of drug candidates and thereby reduce the extremely high attrition rates that plague drug development, often due to unforeseen toxicity (Hay et al., 2014; Ledford, 2011). Cell-based microarrays have been used to screen for the effects of arrayed ECM proteins on neural stem cell proliferation and differentiation (Soen et al., 2006). The experimental platforms for these cell-based studies, however, have been almost uniformly focused on two-dimensional (2D) environments, despite the fact that three-dimensional (3D) approaches have gained increasing interest (Ranga et al., 2014). In particular, 3D culture models may better mimic the in vivo cellular microenvironment, which can be critical in phenotypic screens (Barcellos-Hoff et al., 1989; Yarmush and King, 2009). However, challenges remain in developing and implementing microarray-based 3D systems for screening purposes, including developing stable natural or synthetic matrices that enable rapid diffusion of soluble factors and reagents for 3D-based studies (Li et al., 2014; Ranga et al., 2014; Yarmush and King, 2009). Furthermore, samples arrayed on a surface often share the same culture medium, whereas it would be desirable to screen many liquid media compositions using a 3D culture platform. To address these limitations, we developed a microfabricated plastic “chip” system capable of 3D cell culture at the nanoliter scale. This platform, which has been described previously for rapid toxicity screening of compounds with human hepatocellular carcinoma cells, consists of two complementary chips that “stamp” together to generate up to 532 independent microscale cultures per chip (Kwon et al., 2014).