Veit Bergendahl

Feeder-independent culture of human embryonic stem cells.

We recently reported the development of TeSR1, a serum-free, animal product–free medium that supports the derivation and long-term feeder-independent culture of human embryonic stem cells. Although the derivation of new human embryonic stem cell lines in those defined conditions offered an important proof of principle, the costs of some of the defined components in that culture system made it impractical for everyday research use. Here we describe modifications to the medium (mTeSR1) that include the use of animal-sourced proteins (bovine serum albumin (BSA) and Matrigel) and cloned zebrafish basic fibroblast growth factor (zbFGF). We include a simple protocol that allows purification of up to 100 mg zbFGF in less than three days (Fig. 1), an amount sufficient to make 1,000 l of mTeSR1 medium. The modifications presented here make mTeSR1 practical for routine research use, and the protocols presented are those currently used in our laboratory for standard human embryonic stem cell culture.*Note: In the version of this Protocol initially published, the references were numbered incorrectly. The error has been corrected in the HTML and PDF versions of the article.

Scalable culture and cryopreservation of human embryonic stem cells on microcarriers

As a result of their pluripotency and potential for unlimited self‐renewal, human embryonic stem cells (hESCs) hold tremendous promise in regenerative medicine. An essential prerequisite for the widespread application of hESCs is the establishment of effective and efficient protocols for large‐scale cell culture, storage, and distribution. At laboratory scales hESCs are cultured adherent to tissue culture plates; these culture techniques are labor‐intensive and do not scale to high cell numbers. In an effort to facilitate larger scale hESC cultivation, we investigated the feasibility of culturing hESCs adherent to microcarriers. We modified the surface of Cytodex 3 microcarriers with either Matrigel or mouse embryonic fibroblasts (MEFs). hESC colonies were effectively expanded in a pluripotent, undifferentiated state on both Matrigel‐coated microcarriers and microcarriers seeded with a MEF monolayer. While the hESC expansion rate on MEF‐microcarriers was less than that on MEF‐plates, the doubling time of hESCs on Matrigel‐microcarriers was indistinguishable from that of hESCs expanded on Matrigel‐coated tissue culture plates. Standard hESC cryopreservation methodologies are plagued by poor viability and high differentiation rates upon thawing. Here, we demonstrate that cryopreservation of hESCs adherent to microcarriers in cryovials provides a higher recovery of undifferentiated cells than cryopreservation of cells in suspension. Together, these results suggest that microcarrier‐based stabilization and culture may facilitate hESC expansion and storage for research and therapeutic applications.

Luminescence Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Essential Protein-Protein Interactions in Bacterial RNA Polymerase

ABSTRACT The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled σ70 and an IC5-labeled fragment of the β′ subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

Optical mapping discerns genome wide DNA methylation profiles

BackgroundMethylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification.ResultsA single molecule approach is presented for the discernment of methylation profiles, based on optical mapping. We report results from a series of pilot studies demonstrating the capabilities of optical mapping as a platform for methylation profiling of whole genomes. Optical mapping was used to discern the methylation profile from both an engineered and wild type Escherichia coli. Furthermore, the methylation status of selected loci within the genome of human embryonic stem cells was profiled using optical mapping.ConclusionThe optical mapping platform effectively detects DNA methylation patterns. Due to single molecule detection, optical mapping offers significant advantages over other technologies. This advantage stems from obviation of DNA modification steps, such as bisulfite treatment, and the ability of the platform to assay repeat dense regions within mammalian genomes inaccessible to techniques using array-hybridization technologies.

Studying sigma-core interactions in Escherichia coli RNA polymerase by electrophoretic shift assays and luminescence resonance energy transfer

Publisher Summary This chapter aims to describe electrophoretic mobility shift (EMS) assays and fluorescence resonance energy transfer (FRET) for several reasons. EMS assays are fairly simple and quick to perform with the equipment present in most biologically oriented laboratories. At the same time, they give useful initial information about a protein binding to another protein, DNA, or RNA. EMS assays are based on the change of mobility of a protein during polyacrylamide gel electrophoresis (PAGE) on binding to DNA, RNA, or another protein. Crucial to EMS assays the fact that the procedure involves separating the complex from the unbound binding partner by size and charge differences is discussed. This changes the equilibrium conditions at which initial binding occurs, and thus weak interactions, such as in complexes that have a half-life shorter than the time scale of the separation step, are under-represented, or cannot be detected, as the interaction does not persist throughout the procedure. It focuses on luminescence resonance energy transfer (LRET)-based assays for a homogeneous assay to measure formation of the σ70– β complex. LRET is a recent modification of FRET that uses a lanthanide-based donor fluorophore. The more general term luminescence instead of fluorescence indicates that lanthanide emission is technically not fluorescence.