br Discussion hESCs provide an extraordinary platform for

Discussion hESCs provide an extraordinary platform for disease modeling, especially for developmental disorders (Avior et al., 2016). We decided to use their unique characteristics to model developmental and tumorigenic aspects of pRB and TRb, a severe neonatal malignancy. Using gene editing, we generated heterozygous and homozygous RB1 mutant hESCs (Figures 1A and S1). Although biallelic inactivation of RB1 is believed to be embryonic lethal, RB1-null hESCs survive and proliferate normally in culture. These observations are similar to those provided by mouse models of retinoblastoma (Jacks et al., 1992). The ability of the cells to perform a normal Celecoxib may originate from a functional redundancy of pRB homologous proteins, p107 and p130, encoded by the retinoblastoma-like 1 and 2 (RBL1/2) genes, respectively (Mulligan and Jacks, 1998). Indeed, RB1 cells showed a significant upregulation of RBL1 expression (Figure 1C). Although such an attempt might be sufficient to maintain a normal cell cycle (Figures S1D and S1E), we found that the expression of pRB cofactors and targets is altered in RB1-null hESCs (Figure 1D). pRB cofactor upregulation following pRB ablation could be explained by allowing an autoregulation through positive feedback loops, as shown for E2F family members and TBX22 (Andreou et al., 2007; Johnson et al., 1994), or by ablating pRB-regulated degradation, as shown for EID1 (Miyake et al., 2000). As many pRB cofactors are transcription factors, the downstream effect of their upregulation is alterations in RB1 target expression (Figure 1D). The most substantial difference between control and RB1−/− hESCs resided in their mitochondrial properties. Mutant cells expressed lower levels of mitochondrially transcribed RNA, with a lower mtDNA copy number and a significantly aberrant mitochondrial function (Figures 2 and S2). The latter included a dramatic reduction in oxidative phosphorylation alongside an increased glycolytic rate and elevated basal levels of ROS (Figures 2C and 4D). Proliferation rate in mutant cells did not change despite the decrease in mitochondrial ATP production, probably due to an adequate compensatory increase in glycolytic ATP generation (Figures 2C and S1E). It is noteworthy that MYC, an E2F-pRB target upregulated in mutant cells (Figure 1D), was suggested to take part in glycolysis upregulation in naive pluripotent cells (Gu et al., 2016), and therefore could influence the observed phenotype. TEM micrographs revealed that many of the mitochondria in RB1−/− cells were aberrant, being either elongated, deformed, or undergoing autophagy (ghost mitochondria) (Carta et al., 2000) (Figure 2D). Similar phenotypes were recently shown to characterize poorly differentiated retinoblastoma tumor cells (Singh et al., 2016a). It is currently unknown whether these phenomena are a direct result of RB1 biallelic inactivation. However, there have been reports of pRB localizing to the mitochondria (Ferecatu et al., 2009) and directly affecting mitochondrial-mediated apoptosis (Hilgendorf et al., 2013), perhaps mediating mitochondria biogenesis and function. Together, these data suggest the RB1−/− hESCs have cellular characteristics in common with retinoblastoma tumors, contributing to their use as a disease model and in drug discovery. Utilizing hESC capabilities to differentiate toward the three embryonic germ layers, we used RB1-null hESCs to generate teratomas. These heterogeneous tumors are indicative of hESC tumorigenic potential and shed light on embryonic developmental processes (Avior et al., 2015; Ozolek and Castro, 2011). RB1-null cells generated significantly larger teratomas, suggesting that RB1 inactivation can enhance cell proliferation alongside differentiation. Histologically, RB1 teratomas had a dramatic enhancement of neural structures (Figures 3B and 3C). The neural nature of the tumors echoes the neural component of TRb malignancy, strengthening the validity of our model.