Overall our robust transcriptome data in cells undergoing hi

Overall, our robust transcriptome data in Senexin A undergoing hiPSC reprogramming showed dramatic changes in cell signaling pathways, human-specific AS, and ASE during the progressive cell fate change of fibroblasts to iPSCs (Figure 6E). The data will broaden the knowledge of the reprogramming process and human-specific gene regulation.
Experimental Procedures
Author Contributions
Acknowledgments
Introduction Three-dimensional in vitro models of the human intestine, such as human intestinal organoids (HIOs), offer immense promise for gastrointestinal (GI) research. HIOs offer the unique ability to understand physiologic interactions of the intestine in an in vitro setting and have potential for future applications in tissue engineering and regenerative medicine. Reports include the use of HIOs for studying viral infections (Finkbeiner et al., 2012), bacterial infections (Leslie et al., 2015), inflammatory bowel disease (Xue et al., 2013), and development of enteroendocrine and β cells (Chen et al., 2014; Du et al., 2012). HIOs are generated using directed differentiation of human pluripotent stem cells (hPSCs) in a stepwise differentiation process that mimics embryonic development (Finkbeiner and Spence, 2013; McCracken et al., 2011; Spence et al., 2011; Wells and Spence, 2014). We previously showed that HIOs contain both mesenchymal and epithelial cell populations, including the four major epithelial cell types of the small intestine (enterocytes, goblet, Paneth, and enteroendocrine cells) as well as LGR5+ epithelial cells. Moreover, we demonstrated that some cell types were functional, absorbing peptides (enterocytes) and secreting mucin (goblet cells). These findings suggested that HIOs represent mature, functional intestinal tissue (Spence et al., 2011). This conclusion was in contrast to findings in other hPSC-derived lineages, such as pancreatic β-like cells and in lung organoids, which were shown to be more similar to fetal rather than adult tissue (Dye et al., 2015; Hrvatin et al., 2014). Interestingly, follow-up studies by other groups and ours using HIOs have suggested that HIOs are less mature than adult tissue (Fordham et al., 2013), and HIOs transplanted into the mouse kidney capsule exhibit enhanced cellular function and morphology (Watson et al., 2014). However, the maturity of HIO tissues in vitro remained uncertain, as it was unclear if HIOs represented tissue that was fetal in nature or, alternatively, if expression levels of some genes simply remained low in vitro. Therefore, in this study we sought to do the following: (1) investigate the developmental state of maturity of in-vitro-derived HIOs, (2) determine if HIOs are more similar to human fetal or adult intestinal tissue, and (3) identify cellular and molecular hallmarks of human intestinal maturation. Using RNA-sequencing (RNA-seq) and transcriptome-wide comparisons of HIOs, human fetal, and human adult intestinal tissue, our data show that while HIOs possess markers for functional cell types as originally reported, they are globally more similar to fetal human intestinal tissue than to adult intestine. We also show that the enhanced cellular differentiation and enhanced cellular function after in vivo engraftment of HIOs under the mouse kidney capsule correspond to mature adult-like intestinal tissue. Hallmarks of this maturation process include strong induction of antimicrobial peptide genes produced by Paneth cells and enhanced expression of genes required for digestion. We also found that expression of the Notch-dependent intestinal stem cell (ISC) marker OLFM4 (van der Flier et al., 2009b; VanDussen et al., 2012) is very weakly expressed in HIOs and human fetal tissue, and that acquisition of high levels of OLFM4 expression in the crypt is a hallmark of intestinal maturation. This finding also was confirmed in the developing mouse intestine. Taken together, our results identify major cellular changes that take place during human fetal-to-adult intestinal maturation, demonstrate that acquisition of OLMF4 expression in the crypt is a hallmark of maturation, and highlight that HIOs are a tractable model system that represents human fetal intestinal tissue.