Affiliation: Institute of Genetics and Biophysics "A. Buzzati-Traverso", CNR
Location: Naples, Italy
The Cell-maker: an automation platform for phenotypic screens on Embryonic Stem Cells (ESCs)
ESCs got increasing importance as a model system in basic research, drug discovery and biomedical applications. However, despite their potential, the conventional methods to culture them and in particular to generate ESC-derived somatic progenies are still laborious and poorly reproducible, mainly due to the high variability of manual cell cultures and the limited availability of molecules controlling ESC growth and differentiation. To reverse this trend, the combined automation of the procedures required for both ESC manipulation and molecular screening provides a promising opportunity to identify new regulators of Stem Cell Biology. With this aim, the Cell-maker, a novel integrated robotic platform, has been developed within the Stem Cell Fate Laboratory to fully automate the time-consuming culturing tasks in 96-well microplate format, allowing the screening of small molecules on ESC-based phenotypic assays (proliferation, pluripotency, colony morphology, targeted differentiation).
Metabolites control metastability and pluripotency of mouse Embryonic Stem Cells (mESCs)
The molecular mechanisms controlling the metastability of mESCs, i.e. their capability to fluctuate between different states of pluripotency, are not fully resolved. By exploiting the Cell-maker automation platform for phenotypic screens, we tested a library of metabolites on two different ESC-based assays, proliferation and colony morphology, identifying two metabolically related amino acids, namely L-Proline (L-Pro) and L-Ornithine (L-Orn), as key regulators of ESC metastability. Both compounds, but mainly L-Pro, force ESCs toward a novel Epiblast Stem Cell (EpiSC) - like state, in a dose- and time-dependent manner. Unlike described EpiSCs, L-Pro-induced Cells (PiCs) contribute to chimeric embryos and rely on leukemia inhibitor factor (LIF) to self-renew. Furthermore, PiCs revert to ESCs or differentiate randomly upon removal of either L-Pro or LIF, respectively. Most remarkably, PiC generation depends on both L-Pro metabolism (uptake and oxidation) and Fgf5 induction, and is strongly counteracted by antioxidants, mainly L-Ascorbic acid (Vitamin C). ESCs <-> PiCs phenotypic transition thus represents a previously undefined dynamic equilibrium between pluripotent states, which can be unbalanced either toward an EPiSc-like or ESC phenoptyoe by L-Pro/L-Orn or Vitamin C treatments, respectively. All together, our data provide evidence that ESC metastability can be controlled/regulated at a metabolic level.
Casalino, L., Magnani, D., De Falco, S., Filosa, S., Minchiotti, G., Patriarca, E.J., and De Cesare, D. (2011). An Automated High Throughput Screening-Compatible Assay to Identify Regulators of Stem Cell Neural Differentiation. Mol Biotechnol.
Casalino, L., Comes, S., Lambazzi, G., De Stefano, B., Filosa, S., De Falco, S., De Cesare, D., Minchiotti, G., and Patriarca, E.J.(2011). Control of Embryonic Stem Cell Metastability by L-Proline Catabolism.Journal of Molecular Cell Biology 3, 108-122.
Talotta, F., Mega, T., Bossis, G., Casalino, L., Basbous, J., Jariel-Encontre, I., Piechaczyk, M., and Verde, P. (2010). Heterodimerization with Fra-1 cooperates with the ERK pathway to stabilize c-Jun in response to RAS oncoprotein. Oncogene 29, 4732-4740.
Talotta, F., Cimmino, A., Matarazzo, M., Casalino, L., De Vita, G., DEsposito, M., Di Lauro, R., and Verde, P. (2008). An autoregulatory loop mediated by miR-21 and PDCD4 controles the AP-1 activity in RAS transformation. Oncogene 28, 73-84.
Verde, P., Casalino, L., Talotta, F., Yaniv, M., and Weitzman, J. B. (2007). Deciphering AP-1 function in tumorigenesis: fra-ternizing on target promoters. Cell Cycle 6, 2633-2639.
Casalino, L., Bakiri, L., Talotta, F., Weitzman, J. B., Fusco, A., Yaniv, M., and Verde, P. (2007). Fra-1 promotes growth and survival in RAS-transformed thyroid cells by controlling cyclin A transcription. The EMBO Journal 26, 1878-1890.
Casalino, L., De Cesare, D., and Verde, P. (2003). Accumulation of Fra-1 in ras-transformed cells depends on both transcriptional autoregulation and MEK-dependent posttranslational stabilization. Molecular and Cellular Biology 23, 4401-4415.
- Accumulation of Fra-1 in ras-transformed cells depends on both transcriptional autoregulation and MEK-dependent posttranslational stabilizationLaura Casalino
Institute of Genetics and Biophysics A Buzzati Traverso, Consiglio Nazionale delle Ricerche, 80125 Naples, Italy
Mol Cell Biol 23:4401-15. 2003..These data show that the alteration of multiple regulatory mechanisms is required for the constitutive activation of Fra-1 as a nuclear target of ras transformation...
- Fra-1 promotes growth and survival in RAS-transformed thyroid cells by controlling cyclin A transcriptionLaura Casalino
A Buzzati Traverso Institute of Genetics and Biophysics, CNR, Naples, Italy
EMBO J 26:1878-90. 2007..Fra-1 also induced the expression of JunB, which in turn interacts with the cyclin A promoter. Hence, Fra-1 induction is important in thyroid tumorigenesis, critically regulating cyclin expression and cell-cycle progression...
- Deciphering AP-1 function in tumorigenesis: fra-ternizing on target promotersPasquale Verde
Institute of Genetics and Biophysics A Buzzati Traverso, CNR, Naples, Italy
Cell Cycle 6:2633-9. 2007..Here, we focus on the Fra-1 protein in tumorigenesis, which offers an illustrative example of this helter-skelter voyage...
- An automated high throughput screening-compatible assay to identify regulators of stem cell neural differentiationLaura Casalino
Stem Cell Fate Laboratory, Institute of Genetics and Biophysics A Buzzati Traverso, National Research Council, Naples, Italy
Mol Biotechnol 50:171-80. 2012..Moreover, we performed a pilot screening providing proof of concept that this assay allows the identification of regulators of ESC neural differentiation in full automation...