Supplementary Materials1. Open in a separate window In Brief Zhang et al. display a small-molecule collection and find that pharmacologic inhibition of TGF- and PAK1-ROCK-Myosin II, in low calcium conditions, supports prolonged development of epithelial stem cells in 2D file format. This approach enhances the potential of tissue-resident epithelial stem cells for cell therapy. Intro Tissue-resident stem cells guarantee homeostasis and cells restoration throughout the lifetime of an individual. In various epithelia, the stem and progenitor cells residing in the basal coating are designated by KRT5 and TP63 and have infinite self-renewal ability (Blanpain and (+)-JQ1 inhibition Fuchs, 2014; Donati and Watt, 2015; Hogan et al., 2014; Rock et al., 2010). However, it has been hard to extensively increase epithelial cells in feeder-free condition due to the CDKN2A-dependent stasis (Shay and Wright, 2007). Immortalization using telomerase reverse transcriptase (TERT) or viral genes (SV40T or HPV16 E6/E7) significantly alters epithelial cells behavior, limiting their energy for studying normal biology or as drug-screening models (Miller and Spence, 2017). Lack of suitable long-term development methods offers hampered epithelial stem cell biology study and greatly stalled improvements in regenerative medicine exploiting their potential. Pluripotent stem cells (PSCs), including induced PSCs, have been the subject of intense study in the hope that they offer physiology-relevant models and solutions for regenerative medicine. However, they face difficulties (+)-JQ1 inhibition including donor variability, acquired oncogenic mutations, and inefficient differentiation toward adult cell types (Avior et al., 2016; Merkle et al., 2017). Motivating progress has been made in developing methods that allow continuous propagation of epithelial cells. Liu et al. proposed that feeder cells and Rho-kinase (ROCK) inhibitor Y-27632 conditionally reprogrammed (CR) epithelial cells to proliferate continually (Butler et al., 2016; Chapman et al., 2010; Liu et al., 2012; Suprynowicz et al., 2012). The Stingl group used a similar approach to increase mammary repopulating devices, an indication of the development of mammary epithelial progenitors (Prater et al., 2014). The CR method has garnered interest due to its successful use in expanding patient-derived epithelial cells to identify effective therapy (Crystal et al., 2014; Yuan et al., 2012). Wang et al. (2015) used feeder cells and several small molecules regulating TGF-, WNT, and NOTCH pathways to expand ground-state intestinal stem cells. However, the use of feeder cells complicates the interpretation of signaling events that govern cell proliferation and creates difficulties in meeting regulatory expectation for developing cell therapy products (Lipsitz et al., 2016). The Clevers group offers led the way in developing feeder-free 3D organoids for intestinal stem cells (Sato et al., 2009, 2011), which has later on expanded to epithelial cells from liver, pancreas, and belly (Boj et al., 2015; Huch et al., 2013, 2015). Stem cells, progenitors, and differentiated epithelial cells are present in the organoid, making it a good model for epithelial cell biology. Katsuda et al. (2017) reported the use of small molecules, including Y-27632, A83-01, and CHIR99021, which converted rodent hepatocytes into proliferative bipotent cells; however, it did not work for human being hepatocytes. To develop medium formulations that address aforementioned issues, AGAP1 including security, reproducibility, and scale-up compatibility, we set off to identify small molecules that support long-term epithelial cell development without feeder cells. We found that the combination of (+)-JQ1 inhibition TGF- signaling inhibition, PAK1-ROCK-Myosin II inhibition, and low extracellular [Ca2+] were key parts that transformed traditional culture medium to enable long-term propagation of epithelial cells from numerous tissues. Large single-cell cloning effectiveness and the ability to differentiate into tissue-specific adult epithelial cell types suggested that stem and progenitor cells were preserved during development. Amazingly, the cells retained genome integrity with no tumorigenic mutations after considerable development as assessed by multiple methods including whole-genome sequencing. Progressive changes in DNA methylation panorama were the by-product of long-term tradition and had little impact on overall gene expression profile. RESULTS TGF- Signaling Inhibition and ROCK Inhibition Synergistically Support Long-Term Epithelial Cell Development in the Absence of Feeder Cells As epithelial cells quickly cease proliferation when the feeder cells or Y-27632 are omitted.