It is important to note that although RB

It is important to note that although RB1 inactivation is unique to retinoblastoma initiation, it is functionally inactivated in most human neoplasms (Weinberg, 1995); therefore, utilizing RB1-mutant fgf receptor inhibitor as a drug screen platform could benefit other malignancies as well. In conclusion, we have generated a model for TRb, a tumor-involving developmental disease, using hESCs. Undifferentiated RB1−/− hESCs recapitulate aspects of tumors cells, and mutant cell-derived teratomas resemble TRb tumor composition. Our model sheds light on developmental roles of RB1 as well as on its tumorigenic effects, and can be exploited for future drug discovery.
Experimental Procedures
Author Contributions
Acknowledgments The authors would like to thank Gahl Levy for his help in mitochondria-related assays, Dr. Yael Friedman and Chava Gliksmn from the Bio-Imaging unit for their work with the transmission electron microscope, and Prof. Eran Meshorer for critically reading the manuscript. Y.A. is a Clore Fellow. N.B. is the Herbert Cohn Chair in Cancer Research. This work was partially supported by the Israel Science Foundation (grant number 269/12), by The Rosetrees Trust, and by The Azrieli Foundation.
Introduction Pluripotent stem cells (PSC) are characterized by the ability to self-renew indefinitely and to differentiate into all of the three germ lineages—ectoderm, mesoderm, and endoderm—of the developing embryo. During the early developmental stages, mouse embryonic stem cells (mESC) are derived from pre-implantation embryos (embryonic day 3.5 [E3.5]) and possess the “naive” or “ground” pluripotent state, while mouse epiblast stem cells (EpiSC) are isolated from the post-implantation epiblast [E6.5] and represent a more differentiated state, termed “primed.” Interestingly, mESC and EpiSC can be reciprocally converted into one another, using genetic and/or epigenetic modifiers (Chenoweth et al., 2010). The maintenance of pluripotency is principally regulated by three transcription factors, OCT4, SOX2, and NANOG, which constitute the core pluripotency network. In addition, a growing number of proteins that positively or negatively modulate the function of the core complex have been identified. The importance of the intrinsic pluripotency regulatory network genes is further highlighted by their ability to reprogram somatic cells to induced pluripotent stem cells (iPSC), a cell type resembling ESC. Notably, genes important for stem cell pluripotency are also active in cancers (Hadjimichael et al., 2015), suggesting shared regulatory mechanisms. The promyelocytic leukemia (Pml) gene was first described in the early 1990s as being targeted by a chromosomal translocation t(15;17) in acute promyelocytic leukemia (Guan and Kao, 2015) that produces an oncogenic PML-retinoic acid receptor α (RARα) fusion protein (PML-RARα). PML protein is the key organizer of spherical subnuclear structures, named PML nuclear bodies (PML-NBs), and is expressed in a variety of tissues. Its function has been thoroughly investigated in multiple cells and it is a critical player in DNA repair, apoptosis, senescence, oncogenesis, and cancer progression (Guan and Kao, 2015). However, its role in stem cells had been neglected until recent studies unveiled intriguing findings regarding PML involvement in the hematopoietic stem cell (HSC) asymmetric divisions and maintenance through activating fatty acid oxidation (FAO) (Nakahara et al., 2014). At the same time, recent reports using embryonal carcinoma cells and ESC showed that PML is involved in the activation of Oct4 gene expression (Chuang et al., 2011) and belongs to a transcriptional repressive complex that is associated with OCT4 and NANOG (Liang et al., 2008). Although these findings link PML with important pluripotency regulators, the exact role of PML in ESC functions has yet to be clarified. Here, we addressed the role of PML by investigating the phenotypes of ESC in which the expression of PML was experimentally up- or downregulated. We show that PML ablation impairs ESC self-renewal and pluripotency and promotes the transition from naive to primed-like pluripotent cell state. Moreover, PML depletion upregulates the expression of mesodermal markers and decreases the differentiation toward definitive endoderm. The effect of PML ablation on ESC differentiation can be rescued by TBX3 (a T-box transcription factor) overexpression. Finally, Pml mouse embryonic fibroblasts (MEF) yield significantly fewer iPSC colonies compared with wild-type (WT) MEF, identifying PML as a pivotal mediator of somatic cell reprogramming.