Methods in molecular biology
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Human embryonic stem cells (hESCs) are pluripotent cells derived from the embryo at the blastocyst stage. Their embryonic origin confers upon them the capacity to proliferate indefinitely in vitro while maintaining the capacity to differentiate into a large variety of cell types. ⋯ Consequently, the possibility to expand hESCs in serum-free and in feeder-free culture conditions is becoming a major challenge to deliver the clinical promises of hESCs. Here, we describe the basic principles of growing hESCs in a chemically defined medium (CDM) devoid of serum and feeders.
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Large numbers of diverse small non-coding RNAs have been discovered and characterized in eukaryotic RNA interference pathways. These small RNAs have distinctive characteristics and are associated with Argonaute family proteins to regulate gene expression and genomes at various levels. These small RNAs include the Dicer-dependent group such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), and the Dicer-independent group such as Piwi-interacting RNAs (piRNAs). This review summarizes the various classes of eukaryotic small RNAs and the general knowledge of their characteristics, biogenesis, and functions, with emphasis on some of the recently identified small RNAs.
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G-quadruplexes are noncanonical secondary structures formed in DNA sequences containing consecutive runs of guanines. DNA G-quadruplexes have recently emerged as attractive cancer therapeutic targets. It has been shown that the 3' G-rich single-stranded overhangs of human telomeres can form G-quadruplex structures. ⋯ Nuclear magnetic resonance (NMR) spectroscopy has been shown to be a powerful method in determining the G-quadruplex structures under physiologically relevant conditions. We present the NMR methodology used in our research group for structure determination of G-quadruplexes in solution and their interactions with small molecule compounds. An example of a G-quadruplex structure formed in the human telomere sequence recently solved in our laboratory is used as an example.
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Phosphoproteomics, the systematic study of protein phosphorylation events and cell signaling networks in cells and tissues, is a rapidly evolving branch of functional proteomics. Current phosphoproteomics research provides a large toolbox of strategies and protocols that may assist researchers to reveal key regulatory events and phosphorylation-mediated processes in the cell and in whole organisms. ⋯ We then discuss various tandem mass spectrometry approaches for phosphopeptide sequencing and quantification, and we consider aspects of phosphoproteome data analysis and interpretation. Efficient integration of these stages of phosphoproteome analysis is highly important to ensure a successful outcome of large-scale experiments for studies of phosphorylation-mediated protein regulation.
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Stem cell culture systems that rely on undefined animal-derived components introduce variability to the cultures and complicate their therapeutic use. The derivation of human embryonic stem cells and the development of methods to produce induced pluripotent stem cells combined with their potential to treat human diseases have accelerated the drive to develop xenogenic-free, chemically defined culture systems that support pluripotent self-renewal and directed differentiation. In this chapter, we describe four xeno-free culture systems that have been successful in supporting undifferentiated growth of hPSCs as well as methods for xeno-free subculture and cryopreservation of hPSCs. Each culture system consists of a xeno-free growth medium and xeno-free substratum: (1) TeSR2™ with human recombinant laminin (LN-511); (2) NutriStem™ with LN-511; (3) RegES™ with human foreskin fibroblasts (hFFs); (4) KO-SR Xeno-Free™/GF cocktail with CELLstart™ matrix.