Methods in molecular biology
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Human pluripotent stem cells (PSCs), which include human embryonic stem cells (ESCs) as well as induced pluripotent stem cells (iPSCs), represent an important source of cellular therapies in regenerative medicine and the study of early human development. As such, it is becoming increasingly important to develop methods for the large-scale banking of human PSC lines. There are several well-established methods for the propagation of human PSCs. ⋯ Nevertheless, as the field develops, it will no doubt become increasingly important to produce a bank of cells for clinical use without xenogeneic reagents, particularly nonhuman feeder cells which might harbor viruses with potential risk to human health or cell product integrity. Thus, even for cell lines previously exposed to xenogeneic reagents, it is important to minimize any subsequent exposure of the cell lines to additional adventitious agents. We have specifically described procedures for the growth of hESCs on Matrigel, an animal-matrix, and CELLstart, an animal-free matrix, and these can be used to produce hESCs as part of a clinical manufacturing process.
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Mouse embryonic stem cells (mESCs) were first derived and cultured almost 30 years ago and ever since have been valuable tools for creating knockout mice and for studying early mammalian development. More recently (1998), human embryonic stem cells (hESCs) have been derived from blastocysts, and numerous methods have evolved to culture hESCs in vitro in both complex and defined media. hESCs are especially important at this time as they could potentially be used to treat degenerative diseases and to access the toxicity of new drugs and environmental chemicals. For both human and mouse ESCs, fibroblast feeder layers are often used at some phase in the culturing protocol. ⋯ These basic protocols are intended for researchers wanting to develop stem cell research in their labs. These protocols have been tested in our laboratory and work well. They can be modified and adapted for any relevant user's particular purpose.
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Since the first fungal genome was sequenced in 1996, sequencing technologies have advanced dramatically. In recent years, it has become possible to cost-effectively generate vast amounts of DNA sequence data using a number of cell- and electrophoresis-free sequencing technologies, commonly known as "next" or "second" generation. In this chapter, we present a brief overview of next-generation sequencers that are commercially available now. Their potential applications in fungal genomics studies are discussed.
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Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite for better controlling stem cell differentiation to specific fates. Reversible protein phosphorylation is one of the most important posttranslational modifications regulating signaling pathways in biological processes. Global analysis of dynamic changes in protein phosphorylation is, therefore, key to understanding signaling at the system level. ⋯ Our method combines the use of strong cation exchange (SCX) and titanium dioxide (TiO(2)) for phosphopeptide enrichment, high-resolution MS for peptide and protein identification, and stable isotope labeling by amino acids in cell culture (SILAC) for quantification. This approach allows us to identify thousands of phosphorylation sites and profile their relative abundance during differentiation. This systems-biology-based approach provides new insights into how human pluripotent stem cells exit the pluripotent state.
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For certain applications, particularly experiments involving high-resolution imaging, it is necessary to culture cells on glass slides or cover glasses. This chapter describes techniques for successfully growing human embryonic stem cells (hESCs) on glass surfaces under three different conditions - serum-containing, serum-free, and following single-cell dissociation. It is anticipated that these techniques will extrapolate to other types of pluripotent stem cells such as induced pluripotent stem cells (iPSCs) and embryonic germ cells (EGCs).