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...

A cell-based screen of chemical libraries was carried out to identify small molecules that control the self-renewal of ES cells. A previously uncharacterized heterocycle, SC1, was discovered that allows one to propagate murine ES cells in an undifferentiated, pluripotent state under chemically defined conditions in the absence of feeder cells, serum, and leukemia inhibitory factor. Long-term SC1-expanded murine ES cells can be differentiated into cells of the three primary germ layers in vitro and also can generate chimeric mice and contribute to the germ line in vivo. Biochemical and cellular experiments suggest that SC1 works through dual inhibition of RasGAP and ERK1. Molecules of this kind may not only facilitate practical applications of stem cells in research and therapy, but also provide previously undescribed insights into the complex biology of stem cells.

Many neurological and psychiatric disorders affect the cerebral cortex, and a clearer understanding of the molecular processes underlying human corticogenesis will provide greater insight into such pathologies. To date, knowledge of gene expression changes accompanying corticogenesis is largely based on murine data. Here we present a searchable, comprehensive, temporal gene expression data set encompassing cerebral cortical development from human embryonic stem cells (hESCs). Using a modified differentiation protocol that yields neurons suggestive of prefrontal cortex, we identified sets of genes and long noncoding RNAs that significantly change during corticogenesis and those enriched for disease-associations. Numerous alternatively spliced genes with varying temporal patterns of expression are revealed, including TGIF1, involved in holoprosencephaly, and MARK1, involved in autism. We have created a database (http://cortecon.neuralsci.org/) that provides online, query-based access to changes in RNA expression and alternatively spliced transcripts during human cortical development.

Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus-silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV-1) vectors dominantly silence a linked locus control region (LCR) beta-globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus-transduced embryonic stem (ES) cells are silenced predominantly 3 days post-infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase-null [dnmt3a-/-;dnmt3b-/-] ES cells. MSCV and HIV-1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.