Project 1: Human Pluripotent Stem Cells - A simple, scalable system for drug discovery in the Mdm2, p53, p21 stress response pathway: The capacities of human embryonic stem cells (hESC) for self-renewal in vitro while maintaining the pluripotent state will support new technologies allow to study and treat many severe human diseases. However, the current methods to sustain these cells in culture are inefficient and limit the implementation of this new approach. The central difficulties are (i) the very low plating efficiency when the cells are seeded as single cells (ii) the poor recovery when using cryopreserved cells and (iii) the heterogeneity of cellular states when growing undifferentiated cells. To solve these problems, we have established a robust culture system that allows the cells to plated as single cells and grown in a monolayer. Inhibitors of the Janus activated- (JAK) and Rho- (ROCK) kinases were used to improve the single-cell plating efficiency (SCPE) permitting the maintenance of undifferentiated hESC lines under feeder conditions. Continuous live cell imaging is made possible by this monolayer culture system. To take advantage of this feature, we established a knock-in reporter for the MDM2-p53 stress-response pathway that is involved in the regulation of cell death and is often mutated in human cancer. A fluorescent reporter was incorporated by homologous recombination into the p21 gene allow real-time analysis of stress responses in hESCs under controlled growth conditions. This approach was used to show that JAKi- and ROCKi-mediated survival is achieved by distinct mechanisms demonstrating proof-of-concept for this strategy to study p53 responses in human ES cells. Project 2: Growth control mechanisms in neural stem cells - FGF2 and insulin act by distinct mechanisms to regulate cyclin levels and control growth. Neural stem cells (NSCs) are multipotent cells able to differentiate into neurons, astrocytes, and oligodendrocytes. Current clinical trials assessing the value of NSC based cell therapies point to the importance of understanding the mechanisms controlling their proliferation. The development of serum-free conditions for their growth provides a platform to define the molecular mechanisms triggered by defined growth factors. FGF2 and insulin are essential for the serum-free growth and survival of NSCs. Because NSCs require both factors, it is important to define the molecular mechanism that integrates signals from these two external signals. In this project, we are defining the signaling pathways connecting these ligands to the cell cycle machinery. Our work shows that FGF2 signals through Erk1/2 while insulin exclusively signals through the PI3k/Akt pathway. FGF2 phosphorylation of Erk1/2 leads to the stimulation of cyclin D1 and D2 expression through transcription of c-Fos and c-Jun. In contrast, insulin signals through the PI3k/Akt pathway to increase the level of cyclins D1 and D2 by post-transcriptional modulation. This work suggests that FGF2 and insulin use distinct mechanisms to increase cyclin levels and have complementary effects on cell-cycle progression. This dual dependence could provide spatial and temporal control during fetal brain development and in therapeutic contexts.

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Lathia, J D; Hitomi, M; Gallagher, J et al. (2011) Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions. Cell Death Dis 2:e200
Najm, Fadi J; Chenoweth, Josh G; Anderson, Philip D et al. (2011) Isolation of epiblast stem cells from preimplantation mouse embryos. Cell Stem Cell 8:318-25
McKay, Ron (2011) Developmental biology: Remarkable role for the placenta. Nature 472:298-9
International Stem Cell Initiative Consortium; Akopian, Veronika; Andrews, Peter W et al. (2010) Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell Dev Biol Anim 46:247-58
Androutsellis-Theotokis, Andreas; Rueger, Maria A; Park, Deric M et al. (2010) Angiogenic factors stimulate growth of adult neural stem cells. PLoS One 5:e9414
Chenoweth, Josh G; McKay, Ronald D G; Tesar, Paul J (2010) Epiblast stem cells contribute new insight into pluripotency and gastrulation. Dev Growth Differ 52:293-301
Andrews, Peter W; Benvenisty, Nissim; Knowles, Barbara B et al. (2010) Human ES cell lines--introduction. In Vitro Cell Dev Biol Anim 46:167-8
Androutsellis-Theotokis, Andreas; Walbridge, Stuart; Park, Deric M et al. (2010) Cholera toxin regulates a signaling pathway critical for the expansion of neural stem cell cultures from the fetal and adult rodent brains. PLoS One 5:e10841
Lemaitre, Herve; Mattay, Venkata S; Sambataro, Fabio et al. (2010) Genetic variation in FGF20 modulates hippocampal biology. J Neurosci 30:5992-7
McKay, Ronald D G (2009) Stem cells by the sea. Cell Stem Cell 4:511-2

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