Addition of glucose to quiescent Saccharomyces cerevisiae cells causes the immediate induction of $1000 genes. These genes include ribosomal proteins (RP) and non-RP genes needed for ribosome production and other growth processes. RRPE sequence elements are commonly found 59 of non-RP growth gene ORFs, and Stb3 has recently been identified as an RRPE binding protein. Stb3 overexpression (Stb3OE) produces a slow growth phenotype that is associated with reduced expression of non-RP genes and a drop in the rate of amino acid incorporation. Genes affected by Stb3 are associated with a TGAAAAA motif. Stb3 is restricted to the nucleus in quiescent cells and is immediately released into the cytoplasm after glucose repletion. The Stb3OE slow growth phenotype is reversed by loss of Hos2 histone deactylase activity, consistent with the idea that repression involves histone deacetylation. SCH9 overexpression or PPH22 deletion, mutations that activate target of rapamycin (Tor) nutrient sensing pathways, also reverse the Stb3OE phenotype. Inhibition of Tor signaling makes the phenotype more severe and restricts Stb3 to the nucleus. The results support a model in which Stb3 is one of the components that repress a large set of growth genes as nutrients are depleted. This repression is ended by glucose.

Saccharomyces cerevisiae are able to control growth in response to changes in nutrient availability. The limitation for single macronutrients, including nitrogen (N) and phosphate (P), produces stable arrest in G1/G0. Restoration of the limiting nutrient quickly restores growth. It has been shown that glucose (G) depletion/repletion very rapidly alters the levels of more than 2000 transcripts by at least 2-fold, a large portion of which are involved with either protein production in growth or stress responses in starvation. Although the signals generated by G, N, and P are thought to be quite distinct, we tested the hypothesis that depletion and repletion of any of these three nutrients would affect a common core set of genes as part of a generalized response to conditions that promote growth and quiescence. We found that the response to depletion of G, N, or P produced similar quiescent states with largely similar transcriptomes. As we predicted, repletion of each of the nutrients G, N, or P induced a large (501) common core set of genes and repressed a large (616) common gene set. Each nutrient also produced nutrient-specific transcript changes. The transcriptional responses to each of the three nutrients depended on cAMP and, to a lesser extent, the TOR pathway. All three nutrients stimulated cAMP production within minutes of repletion, and artificially increasing cAMP levels was sufficient to replicate much of the core transcriptional response. The recently identified transceptors Gap1, Mep1, Mep2, and Mep3, as well as Pho84, all played some role in the core transcriptional responses to N or P. As expected, we found some evidence of cross talk between nutrient signals, yet each nutrient sends distinct signals.

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Heart failure is the leading cause of death in the western world and as such, there is a great need for new therapies. Heart failure has a variable presentation in patients and a complex etiology; however, it is fundamentally a condition that affects the mechanics of cardiac contraction, preventing the heart from generating sufficient cardiac output under normal operating pressures. One of the major issues hindering the development of new therapies has been difficulties in developing appropriate in vitro model systems of human heart failure that recapitulate the essential changes in cardiac mechanics seen in the disease. Recent advances in stem cell technologies, genetic engineering, and tissue engineering have the potential to revolutionize our ability to model and study heart failure in vitro. Here, we review how these technologies are being applied to develop personalized models of heart failure and discover novel therapeutics.

Background Luminescent reporter proteins are vital tools for visualizing cells and cellular activity. Among the current toolbox of bioluminescent systems, only bacterial luciferase has genetically defined luciferase and luciferin synthesis pathways that are functional at the mammalian cell temperature optimum of 37 °C and have the potential for in vivo applications. However, this system is not functional in all cell types, including stem cells, where the ability to monitor continuously and in real-time cellular processes such as differentiation and proliferation would be particularly advantageous. Results We report that artificial subdivision of the bacterial luciferin and luciferase pathway subcomponents enables continuous or inducible bioluminescence in pluripotent and mesenchymal stem cells when the luciferin pathway is overexpressed with a 20–30:1 ratio. Ratio-based expression is demonstrated to have minimal effects on phenotype or differentiation while enabling autonomous bioluminescence without requiring external excitation. We used this method to assay the proliferation, viability, and toxicology responses of iPSCs and showed that these assays are comparable in their performance to established colorimetric assays. Furthermore, we used the continuous luminescence to track stem cell progeny post-differentiation. Finally, we show that tissue-specific promoters can be used to report cell fate with this system. Conclusions Our findings expand the utility of bacterial luciferase and provide a new tool for stem cell research by providing a method to easily enable continuous, non-invasive bioluminescent monitoring in pluripotent cells.