EGFP-positive cells were determined by flow cytometry after drug selection

EGFP-positive cells were determined by flow cytometry after drug selection. auto- and reciprocal activations at the transcription level, which is usually believed to be responsible for the maintenance of human embryonic stem cell (hESC) pluripotency (Boyer et?al., 2005). At the same time, multiple protein factors belonging to a diversity of functional groups, such as transcription PSMA617 TFA factors, epigenetic factors, and signaling components, work cooperatively to form an expanded pluripotency factor network that supports the core pluripotency network (Boyer et?al., 2005). In contrast to the well-defined core network, our knowledge of this expanded pluripotency network, including its components, the interactions between these components, and the mechanism of interaction between the expanded network and the core network, remains insufficient. Bioactive small molecules have been applied to the field of hESC research with success. Many such studies have applied small molecules as modulators of lineage-specific differentiations (Borowiak et?al., 2009, Chen et?al., 2009, Chen et?al., 2012, Gonzalez et?al., 2011a, Lian et?al., 2012, Mahmood et?al., 2010). Other studies have exploited small molecules as chemical probes to uncover novel molecular mechanisms underlying hESC pluripotency or differentiation (Chen et?al., 2006, Xu et?al., 2010, Zhu et?al., 2009). High-throughput screenings (HTS) were usually conducted for the search of such molecules. If the mechanism of action was unknown for a Rabbit Polyclonal to NEIL1 given molecule, affinity-based target identification methods can be used to identify its?biological target(s). These studies have been used to identify novel protein factors and to unveil previously unknown molecular mechanisms that regulate hESC fate determination (Xu et?al., 2008). PSMA617 TFA In recent years, hESCs and human induced pluripotent stem cells (hiPSCs) have been used successfully for HTS in several studies (Barbaric et?al., 2010, Ben-David et?al., 2013, Desbordes et?al., 2008, Gonzalez et?al., 2011b, Kameoka et?al., 2014, Kumagai et?al., 2013, Manganelli et?al., 2014, Xu et?al., 2010). However, the high cost associated with the maintenance and scale-up of human pluripotent stem cells (hPSCs) inevitably limits the level of their application in HTS studies. We chose to explore an alternative source of pluripotent stem cells, human embryonal carcinoma cells (hECCs), as a robust platform for HTS with low cost. hECCs are pluripotent stem cells derived from human teratocarcinomas and are considered to be the malignant counterparts of hESCs. The molecular regulatory mechanism of hECC pluripotency has been shown to mimic that of hESCs (Josephson et?al., 2007). Because of their cancerous nature, hECCs are not prone to spontaneous differentiation and require a less demanding culture condition compared with hPSCs. Experimental results acquired from studies using hECCs have been proven to be highly stable and readily reproducible (Josephson et?al., 2007), making hECCs ideal candidate platforms for HTS purposes. Based on the concept of hECC-based HTS, we established a pluripotency reporter system using the hECC line NTERA-2. Using this system, we conducted a large-scale chemical screening and found 122 small molecules that disrupt hESC pluripotency. One of these molecules, which we named Displurigen, potently disrupts hESC pluripotency by targeting heat shock 70-kDa protein 8 (HSPA8, the constitutively expressed member of the 70-kDa heat shock protein family), as discovered using affinity-based target identification methods and functional validations. We demonstrated that HSPA8 helps maintain pluripotency by direct binding to the OCT4 protein and facilitating OCT4 binding to DNA. Results Establishment of an NTERA-2 Cell-Based Pluripotency Reporter System To avoid high experimental variations associated with hESC-based HTS platforms, we established an HTS platform using the hECC line NTERA-2. NTERA-2 is a clonal subline of TERA-2, one of the first established hECC lines (Andrews et?al., 1984). NTERA-2 cells are able to differentiate into all three germ layers in?vivo in the form of teratocarcinomas. In culture, these cells differentiate in response to several inducers of differentiation, most PSMA617 TFA notably retinoic acid (RA) (Andrews, 1984), hexamethylene bis-acetamide (HMBA) (Andrews et?al., 1986, Andrews et?al., 1990), and bone morphogenetic protein 7 (Andrews et?al., 1994). These agents PSMA617 TFA also induce differentiation of hESCs (Draper et?al., 2002, Xu et?al., 2002, zur Nieden et?al., 2005). Furthermore, basic fibroblast growth factor (bFGF) helps maintain the pluripotent state of both NTERA-2 cells and hESCs (Andrews et?al., 1984, Thomson et?al., 1998). The similarities in their responses toward external factors indicate that NTERA-2 cells and hESCs have very similar molecular mechanisms governing their respective pluripotent states. An NTERA-2 cell-based reporter cell line was constructed by stable integration of an EGFP reporter gene driven by a 4-kb OCT4 promoter.