Metabolites offer an important unexplored complement to understanding the pluripotency of stem cells. Using mass spectrometry-based metabolomics, we show that embryonic stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. By monitoring the reduced and oxidized glutathione ratio as well as ascorbic acid levels, we demonstrate that the stem cell redox status is regulated during differentiation. Based on the oxidative biochemistry of the unsaturated metabolites, we experimentally manipulated specific pathways in embryonic stem cells while monitoring the effects on differentiation. Inhibition of the eicosanoid signaling pathway promoted pluripotency and maintained levels of unsaturated fatty acids. In contrast, downstream oxidized metabolites (e.g., neuroprotectin D1) and substrates of pro-oxidative reactions (e.g., acyl-carnitines), promoted neuronal and cardiac differentiation. We postulate that the highly unsaturated metabolome sustained by stem cells makes them particularly attuned to differentiate in response to in vivo oxidative processes such as inflammation.

Chemically defined medium (CDM) conditions for controlling human embryonic stem cell (hESC) fate will not only facilitate the practical application of hESCs in research and therapy but also provide an excellent system for studying the molecular mechanisms underlying self-renewal and differentiation, without the multiple unknown and variable factors associated with feeder cells and serum. Here we report a simple CDM that supports efficient self-renewal of hESCs grown on a Matrigel-coated surface over multiple passages. Expanded hESCs under such conditions maintain expression of multiple hESC-specific markers, retain the characteristic hESC morphology, possess a normal karyotype in vitro, as well as develop teratomas in vivo. Additionally, several growth factors were found to selectively induce monolayer differentiation of hESC cultures toward neural, definitive endoderm͞pancreatic and early cardiac muscle cells, respectively, in our CDM conditions. Therefore, this CDM condition provides a basic platform for further characterization of hESC self-renewal and directed differentiation, as well as the development of novel therapies.chemically defined medium E mbryonic stem cells (ESCs), typically derived from the inner cell mass of the blastocyst, can be propagated indefinitely and differentiated into all of the cell types of the embryo proper (1). ESCs not only hold considerable promise for the treatment of a number of devastating diseases but also provide an excellent system for studying early development and human diseases. Human and mouse ESCs are conventionally maintained in culture with feeder cells and͞or mixtures of exogenous factors (1, 2, 12). However, unknown factors secreted from the feeder cells or contained in the serum may have undesired activities (e.g., inducing differentiation or cell death), which require the addition of other factors that inhibit their effects. Such undefined culture conditions is the main source of inconsistency in large scale and long-term expansion of undifferentiated ESCs. Consequently, the establishment of a well defined culture condition for ESCs would facilitate practical applications of human ESCs (hESCs) and allow for studying and controlling signaling inputs that regulate self-renewal or differentiation of ESCs without multiple unknown and variable factors.Recent studies have shown that feeder-fibroblast conditioned medium (CM) (3), high concentrations of basic FGF (bFGF, 100 ng͞ml) (4) and combinations of bFGF with Noggin (4, 5) or TGF-␤͞activin͞Nodal signaling molecules (6-8) can support longterm culture of hESCs grown on an extracellular matrix (ECM)-coated surface (e.g., Matrigel) in feeder-free conditions. However, these medium conditions typically contain a proprietary serum replacement product, which is a complex mixture of many unknown factors with varying batch-to-batch activities. To come one step closer to a completely defined, animal product-free condition (i.e., chemically defined media and ECM) for long-term self-renewal and efficient clonal expansion of hES...

The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi) has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA) with homology to endogenous genes in Caenorhabditis elegans (C elegans), RNAi technology has been adapted to various high-throughput screens (HTS) for genome-wide loss-of-function (LOF) analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.

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