Andrew E. Rodda

Mitochondrial DNA haplotypes define gene expression patterns in pluripotent and differentiating embryonic stem cells.

Mitochondrial DNA haplotypes are associated with various phenotypes, such as altered susceptibility to disease, environmental adaptations, and aging. Accumulating evidence suggests that mitochondrial DNA is essential for cell differentiation and the cell phenotype. However, the effects of different mitochondrial DNA haplotypes on differentiation and development remain to be determined. Using embryonic stem cell lines possessing the same Mus musculus chromosomes but harboring one of Mus musculus, Mus spretus, or Mus terricolor mitochondrial DNA haplotypes, we have determined the effects of different mitochondrial DNA haplotypes on chromosomal gene expression, differentiation, and mitochondrial metabolism. In undifferentiated and differentiating embryonic stem cells, we observed mitochondrial DNA haplotype‐specific expression of genes involved in pluripotency, differentiation, mitochondrial energy metabolism, and DNA methylation. These mitochondrial DNA haplotypes also influenced the potential of embryonic stem cells to produce spontaneously beating cardiomyocytes. The differences in gene expression patterns and cardiomyocyte production were independent of ATP content, oxygen consumption, and respiratory capacity, which until now have been considered to be the primary roles of mitochondrial DNA. Differentiation of embryonic stem cells harboring the different mitochondrial DNA haplotypes in a 3D environment significantly increased chromosomal gene expression for all haplotypes during differentiation. However, haplotype‐specific differences in gene expression patterns were maintained in this environment. Taken together, these results provide significant insight into the phenotypic consequences of mitochondrial DNA haplotypes and demonstrate their influence on differentiation and development. We propose that mitochondrial DNA haplotypes play a pivotal role in the process of differentiation and mediate the fate of the cell. STEM CELLS 2013;31:703–716

Surface grafted poly(ε-caprolactone) prepared using organocatalysed ring-opening polymerisation followed by SI-ATRP

The controlled ring-opening polymerisation (ROP) of an e-caprolactone derivative that contains an ATRP initiator pendant to the ring, γ-(2-bromo-2-methyl propionyl)-e-caprolactone (γ-BMPCL), and its copolymerisation with e-caprolactone (CL) is reported. Functional PCL copolymers that contained pendant ATRP initiators were obtained with higher than previously reported molecular weights using diphenyl phosphate (DPP) as the catalyst at room temperature. Surface-initiated ATRP grafting of oligo(ethylene glycol) methacrylate was successfully carried out on the surface of two dimensional (2D) substrates comprising thin films of a functional PCL copolymer.

Optimization of aqueous SI-ATRP grafting of poly(oligo(ethylene glycol) methacrylate) brushes from benzyl chloride macroinitiator surfaces

Poly(oligo(ethylene glycol) methacrylate) (pOEGMA) brushes were grafted via surface-initiated atom transfer radical polymerization (SI-ATRP) from a poly(styrene-co-vinylbenzyl chloride) macroinitiator. While bromoisobutyryl initiator groups are most commonly used for this purpose, benzyl chloride initiators may be advantageous for some applications due to superior stability. Water-only graft solutions produced thicker brush coatings with superior low fouling properties (low protein adsorption and cell adhesion) versus mixed water/alcohol solutions. Coatings produced using 475 Da OEGMA (methyl ether terminated) further reduced non-specific interactions compared to 360 Da OEGMA (hydroxyl terminated). Initiator density had minimal effect on low fouling properties.

Low fouling electrospun scaffolds with clicked bioactive peptides for specific cell attachment

While electrospun fibers are of interest as scaffolds for tissue engineering applications, nonspecific surface interactions such as protein adsorption often prevent researchers from controlling the exact interactions between cells and the underlying material. In this study we prepared electrospun fibers from a polystyrene-based macroinitiator, which were then grafted with polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP). These brush coatings incorporated a trimethylsilyl-protected PEG-alkyne monomer, allowing azide functional molecules to be covalently attached, while simultaneously reducing nonspecific protein adsorption on the fibers. Cells were able to attach and spread on fibrous substrates functionalized with a pendant RGD-containing peptide, while spreading was significantly reduced on nonfunctionalized fibers and those with the equivalent RGE control peptide. This effect was observed both in the presence and absence of serum in the culture media, indicating that protein adsorption on the fibers was minimal and cell adhesion within the fibrous scaffold was mediated almost entirely through the cell-adhesive RGD-containing peptide.

Controlling integrin-based adhesion to a degradable electrospun fibre scaffold via SI-ATRP

While polycaprolactone (PCL) and similar polyesters are commonly used as degradable scaffold materials in tissue engineering and related applications, non-specific adsorption of environmental proteins typically precludes any control over the signalling pathways that are activated during cell adhesion to these materials. Here we describe the preparation of PCL-based fibres that facilitate cell adhesion through well-defined pathways while preventing adhesion via adsorbed proteins. Surface-initiated atom transfer radical polymerisation (SI-ATRP) was used to graft a protein-resistant polymer brush coating from the surface of fibres, which had been electrospun from a brominated PCL macroinitiator. This coating also provided alkyne functional groups for the attachment of specific signalling molecules via the copper-mediated azide-alkyne click reaction; in this case, a cyclic RGD peptide with high affinity for αvβ3 integrins. Mesenchymal stem cells were shown to attach to the fibres via the peptide, but did not attach in its absence, nor when blocked with soluble peptide, demonstrating the effective control of cell adhesion pathways.

Extending Circulating Tumor DNA Analysis to Ultralow Abundance Mutations: Techniques and Challenges

Liquid biopsies that analyze circulating tumor DNA (ctDNA) hold great promise in the guidance of clinical treatment for various cancers. However, the innate characteristics of ctDNA make it a difficult target: ctDNA is highly fragmented, and found at very low concentrations, both in absolute terms and relative to wildtype species. Clinically relevant target sequences often differ from the wildtype species by a single DNA base pair. These characteristics make analyzing mutant ctDNA a uniquely difficult process. Despite this, techniques have recently emerged for analyzing ctDNA, and have been used in pilot studies that showed promising results. These techniques each have various drawbacks, either in their analytical capabilities or in practical considerations, which restrict their application to many clinical situations. Many of the most promising potential applications of ctDNA require assay characteristics that are not currently available, and new techniques with these properties could have benefits in companion diagnostics, monitoring response to treatment and early detection. Here we review the current state of the art in ctDNA detection, with critical comparison of the analytical techniques themselves. We also examine the improvements required to expand ctDNA diagnostics to more advanced applications and discuss the most likely pathways for these improvements.

Electrospun scaffolds for neural tissue engineering

Abstract Regeneration of the brain following injury is often slow or ineffective due to a dearth of cellular cues that support and guide neural regeneration. Scaffolds have been actively investigated for new therapies targeting neurodegenerative diseases and traumatic brain injury. Electrospinning offers many advantages for the fabrication of a diverse and tunable range of neural scaffolds. The architecture of electrospun scaffolds can be tailored to meet specific requirements such as the culture of different types of neural cells and the incorporation and delivery of bioactive molecules. Electrospun fibers have been highly successful in providing both biological and physical cues to repair the injured brain. This chapter provides an overview of the design principles of electrospun scaffolds for neural tissue engineering, with a focus on brain repair.

Fast femtosecond laser ablation for efficient cutting of sintered alumina and quartz substrates

Ultrafast laser machining of ceramic and crystalline substrates offers many benefits versus mechanical dicing. We optimized femtosecond laser parameters for cutting industry sintered alumina and quartz wafers, yielding drastic improvements in cutting speed and quality.