hESC differentiation can shed light on developmental

hESC differentiation can shed light on developmental and malignant processes. Neural progenitor cells derived from RB1-null hESCs did not differ significantly from control cells (Figure S3A), suggesting that a more complex differentiation paradigm is required to model pRB\'s tumorigenic and developmental roles. We therefore generated teratomas from RB1-null and control cells, allowing us to study the effects of RB1 ablation in vivo. Teratomas derived from RB1−/− hESCs were significantly larger than control tumors (Figures 3A and S3B). Serial sectioning of the teratomas revealed a dramatic expansion of neural structures in tumors derived from RB1−/− clones, visualized by H&E staining and immunofluorescence staining for the specific neuronal marker neural cell adhesion molecule 1 (NCAM1) (Figures 3B and 3C). RNA-seq analysis of the tumors showed a clear separation between expression patterns, with 8.0% (1,545/20,690) of the expressed transcripts at least 3-fold upregulated or downregulated in teratomas derived from RB1−/− clones (Figure S3C). In accordance with the high abundance of neural structures in the RB1−/− tumors, the upregulated genes were significantly enriched for annotations related to the nervous system and neural development (Figure 3D). Downregulated genes were enriched for epidermal related annotations, such as keratinocyte differentiation (Figure 3D). This is intriguing, as both the nervous system and the epidermis are derivatives of the same embryonic germ layer, the ectoderm. To a lesser extent, downregulated genes were enriched for muscle and bone formation categories. Interestingly, both upregulated and downregulated gene groups were enriched for transcripts annotated to include nf-κb pathway to the zinc finger E-box binding homeobox 1 (ZEB1) transcription factor (Figure 3E), which is a master regulator of the epithelial-mesenchymal transition (EMT) (Eger et al., 2005). ZEB1 expression was previously shown to be regulated by pRB and E2F (Liu et al., 2007), and was significantly upregulated in RB1−/− teratomas (Figure 3F). Higher ZEB1 expression correlated with epithelial and mesenchymal marker up- and downregulation, respectively (Figure 3G). ZEB1 target genes downregulated in RB1−/− teratomas included epidermal keratins, while upregulated ZEB1 target genes included neural markers such as NCAM1 and NCAM2, and NEUROD4 (Figures 3H and 3I). Furthermore, ZEB1 was previously shown to promote cell proliferation through regulation of genes such as MKI67 and OLIG2, whose expression was upregulated in RB1−/− teratomas (Figure 3J). Several E2F genes were also upregulated in the mutant teratomas, correlating with enlarged tumors (Figure S3D). Finally, using immunofluorescence staining we found that ZEB1 expression was localized to the same neural structures enlarged following RB1 mutation, suggesting its involvement in this phenotype (Figure 3K). Following the characterization of RB1-null cells and their tumorigenic capacities, we utilized them in a drug-screening platform. As TRb patients are commonly treated with chemotherapies aimed at reducing or eliminating tumors (Dunkel et al., 2010; Wright et al., 2010), we used our disease model to analyze the relative potency of a variety of chemotherapies. An initial drug screening of 119 chemotherapies approved by the Food and Drug Administration (FDA) was performed on a control and RB1-null cell line, with viability assessment 24, 48, and 72 hr after exposure (Figure 4A). A primary analysis was used to identify significant differential effects between the cell lines, and the effect of 12 of the chemotherapies was then analyzed on additional control and mutant cell lines (Figure 4A). The screening revealed carboplatin as a potent chemotherapy with a significant and consistent differential effect, under which the RB1−/− cells showed an elevated sensitivity (Figures 4B and 4C). Several chemotherapies, including carboplatin, are currently used to treat retinoblastoma, but none of the other chemotherapies showed such a specific effect on RB1-null cells (Figures 4B and 4C). Previous reports suggested that platinum-based chemotherapies similar to carboplatin could affect cells through mitochondrial reactive oxygen species (ROS) response (Marullo et al., 2013). Fluorescence-activated cell sorting of cells stained with mitochondrial superoxide indicator following 24 hr of exposure to carboplatin revealed that RB1−/− cells had significantly elevated basal levels of ROS compared with control cells (Figure 4D). Consequently, exposure to carboplatin drives ROS levels in RB1−/− cells dramatically higher, thus suggesting that carboplatin\'s specific effect on RB1−/− cells could originate from their deficient mitochondria. Together, these results demonstrate the potential of our model for identifying compounds specifically targeting RB1-null cells.