Jon P. Golding

Muscle satellite cells adopt divergent fates a mechanism for self-renewal?

Growth, repair, and regeneration of adult skeletal muscle depends on the persistence of satellite cells: muscle stem cells resident beneath the basal lamina that surrounds each myofiber. However, how the satellite cell compartment is maintained is unclear. Here, we use cultured myofibers to model muscle regeneration and show that satellite cells adopt divergent fates. Quiescent satellite cells are synchronously activated to coexpress the transcription factors Pax7 and MyoD. Most then proliferate, down-regulate Pax7, and differentiate. In contrast, other proliferating cells maintain Pax7 but lose MyoD and withdraw from immediate differentiation. These cells are typically located in clusters, together with Pax7−ve progeny destined for differentiation. Some of the Pax7+ve/MyoD−ve cells then leave the cell cycle, thus regaining the quiescent satellite cell phenotype. Significantly, noncycling cells contained within a cluster can be stimulated to proliferate again. These observations suggest that satellite cells either differentiate or switch from terminal myogenesis to maintain the satellite cell pool.

Neural and mammary gland defects in ErbB4 knockout mice genetically rescued from embryonic lethality

Mice lacking the epidermal growth factor receptor family member ErbB4 exhibit defects in cranial neural crest cell migration but die by embryonic day 11 because of defective heart development. To examine later phenotypes, we rescued the heart defects in ErbB4 mutant mice by expressing ErbB4 under a cardiac-specific myosin promoter. Rescued ErbB4 mutant mice reach adulthood and are fertile. However, during pregnancy, mammary lobuloalveoli fail to differentiate correctly and lactation is defective. Rescued mice also display aberrant cranial nerve architecture and increased numbers of large interneurons within the cerebellum.

Defects in pathfinding by cranial neural crest cells in mice lacking the neuregulin receptor ErbB4

Mouse embryos with a loss-of-function mutation in the gene encoding the receptor tyrosine kinase ErbB4 exhibit misprojections of cranial sensory ganglion afferent axons. Here we analyse ErbB4-deficient mice, and find that morphological differences between wild-type and mutant cranial ganglia correlate with aberrant migration of a subpopulation of hindbrain-derived cranial neural crest cells within the paraxial mesenchyme environment. In transplantation experiments using new grafting techniques in cultured mouse embryos, we determine that this phenotype is non-cell-autonomous: wild-type and mutant neural crest cells both migrate in a pattern consistent with the host environment, deviating from their normal pathway only when transplanted into mutant embryos. ErbB4 signalling events within the hindbrain therefore provide patterning information to cranial paraxial mesenchyme that is essential for the proper migration of neural crest cells.

Alignment of Astrocytes Increases Neuronal Growth in Three-Dimensional Collagen Gels and Is Maintained Following Plastic Compression to Form a Spinal Cord Repair Conduit

After injury to the spinal cord, reactive astrocytes form a glial scar consisting of highly ramified cell processes that constitute a major impediment to repair, partly due to their lack of orientation and guidance for regenerating axons. In some nonmammalian vertebrates, successful central nervous system regeneration is attributed to the alignment of reactive glia, which guide axons across the lesion site. Here, a three-dimensional mammalian cell-seeded collagen gel culture system was used to explore the effect of astrocyte alignment on neuronal growth. Astrocyte alignment was mapped within tethered rectangular gels and was significantly greater at the edge and middle of the gels compared to the control unaligned regions. When neurons were seeded on and within astrocyte gels, neurite length was greatest in the areas of astrocyte alignment. There was no difference in expression of astrocyte reactivity markers between aligned and control areas. Having established the potential utility of astrocyte alignment, the aligned gels were plastic compressed, transforming them into mechanically robust implantable devices. After compression, astrocytes remained viable and aligned and supported neurite outgrowth, yielding a novel method for assembling aligned cellular constructs suitable for tissue engineering and highlighting the importance of astrocyte alignment as a possible future therapeutic intervention for spinal cord repair.

Engineered neural tissue with aligned, differentiated adipose-derived stem cells promotes peripheral nerve regeneration across a critical sized defect in rat sciatic nerve.

Adipose-derived stem cells were isolated from rats and differentiated to a Schwann cell-like phenotype in vitro. The differentiated cells (dADSCs) underwent self-alignment in a tethered type-1 collagen gel, followed by stabilisation to generate engineered neural tissue (EngNT-dADSC). The pro-regenerative phenotype of dADSCs was enhanced by this process, and the columns of aligned dADSCs in the aligned collagen matrix supported and guided neurite extension in vitro. EngNT-dADSC sheets were rolled to form peripheral nerve repair constructs that were implanted within NeuraWrap conduits to bridge a 15 mm gap in rat sciatic nerve. After 8 weeks regeneration was assessed using immunofluorescence imaging and transmission electron microscopy and compared to empty conduit and nerve graft controls. The proportion of axons detected in the distal stump was 3.5 fold greater in constructs containing EngNT-dADSC than empty tube controls. Our novel combination of technologies that can organise autologous therapeutic cells within an artificial tissue construct provides a promising new cellular biomaterial for peripheral nerve repair.

Homing of stem cells to sites of inflammatory brain injury after intracerebral and intravenous administration: a longitudinal imaging study

IntroductionThis study aimed to determine the homing potential and fate of epidermal neural crest stem cells (eNCSCs) derived from hair follicles, and bone marrow-derived stem cells (BMSCs) of mesenchymal origin, in a lipopolysaccharide (LPS)-induced inflammatory lesion model in the rat brain. Both eNCSCs and BMSCs are easily accessible from adult tissues by using minimally invasive procedures and can differentiate into a variety of neuroglial lineages. Thus, these cells have the potential to be used in autologous cell-replacement therapies, minimizing immune rejection, and engineered to secrete a variety of molecules.MethodsBoth eNCSCs and BMSCs were prelabeled with iron-oxide nanoparticles (IO-TAT-FITC) and implanted either onto the corpus callosum in healthy or LPS-lesioned animals or intravenously into lesioned animals. Both cell types were tracked longitudinally in vivo by using magnetic resonance imaging (MRI) for up to 30 days and confirmed by postmortem immunohistochemistry.ResultsTransplanted cells in nonlesioned animals remained localized along the corpus callosum. Cells implanted distally from an LPS lesion (either intracerebrally or intravenously) migrated only toward the lesion, as seen by the localized MRI signal void. Fluorescence microscopy of the FITC tag on the nanoparticles confirmed the in vivo MRI data,ConclusionsThis study demonstrated that both cell types can be tracked in vivo by using noninvasive MRI and have pathotropic properties toward an inflammatory lesion in the brain. As these cells differentiate into the glial phenotype and are derived from adult tissues, they offer a viable alternative autologous stem cell source and gene-targeting potential for neurodegenerative and demyelinating pathologies.

Gold nanoparticles for cancer radiotherapy: a review

Radiotherapy is currently used in around 50% of cancer treatments and relies on the deposition of energy directly into tumour tissue. Although it is generally effective, some of the deposited energy can adversely affect healthy tissue outside the tumour volume, especially in the case of photon radiation (gamma and X-rays). Improved radiotherapy outcomes can be achieved by employing ion beams due to the characteristic energy deposition curve which culminates in a localised, high radiation dose (in form of a Bragg peak). In addition to ion radiotherapy, novel sensitisers, such as nanoparticles, have shown to locally increase the damaging effect of both photon and ion radiation, when both are applied to the tumour area. Amongst the available nanoparticle systems, gold nanoparticles have become particularly popular due to several advantages: biocompatibility, well-established methods for synthesis in a wide range of sizes, and the possibility of coating of their surface with a large number of different molecules to provide partial control of, for example, surface charge or interaction with serum proteins. This gives a full range of options for design parameter combinations, in which the optimal choice is not always clear, partially due to a lack of understanding of many processes that take place upon irradiation of such complicated systems. In this review, we summarise the mechanisms of action of radiation therapy with photons and ions in the presence and absence of nanoparticles, as well as the influence of some of the core and coating design parameters of nanoparticles on their radiosensitisation capabilities.

Antioxidant inhibitors potentiate the cytotoxicity of photodynamic therapy

Photodynamic therapy (PDT) is an increasingly popular anticancer treatment that uses photosensitizer, light and tissue oxygen to generate cytotoxic reactive oxygen species (ROS) within illuminated cells. Acting to counteract ROS‐mediated damage are various cellular antioxidant pathways. In this study, we combined PDT with specific antioxidant inhibitors to potentiate PDT cytotoxicity in MCF‐7 cancer cells. We used disulphonated aluminium phthalocyanine photosensitizer plus various combinations of the antioxidant inhibitors: diethyl‐dithiocarbamate (DDC, a Cu/Zn‐SOD inhibitor), 2‐methoxyestradiol (2‐ME, a Mn‐SOD inhibitor), l‐buthionine sulfoximine (BSO, a glutathione synthesis inhibitor) and 3‐amino‐1,2,4‐triazole (3‐AT, a catalase inhibitor). BSO, singly or in combination with other antioxidant inhibitors, significantly potentiated PDT cytotoxicity, corresponding with increased ROS levels and apoptosis. The greatest potentiation of cell death over PDT alone was seen when cells were preincubated for 24 h with 300 μm BSO plus 10 mm 3‐AT (1.62‐fold potentiation) or 300 μm BSO plus 1 μm 2‐ME (1.52‐fold), or with a combination of all four inhibitors (300 μm BSO, 10 mm 3‐AT, 1 μm 2‐ME and 10 μm DDC: 1.4‐fold). As many of these inhibitors have already been clinically tested, this work facilitates future in vivo studies.

ErbB4 Signaling During Breast and Neural Development Novel Genetic Models Reveal Unique ErbB4 Activities

The erbB4 gene encodes one of the four members of the mammalian ErbB family of transmembrane tyrosine kinases. The ErbB4 protein plays a role as a receptor for the neuregulins, a large group of structurally related molecules and a few other epidermal growth factor (EGF)-related polypeptides, such as heparin-binding EGF, betacellulin and epiregulin. The importance of this receptor tyrosine kinase in development has been demonstrated by the generation of mice with a targeted inactivation of the erbB4 gene. Such mice die by embryonic day eleven due to defective trabeculation in the heart, precluding analysis of phenotypes at later stages in development and in the adult. Now, using two unique genetic approaches our laboratories succeeded in overcoming this obstacle. In the first approach, the heart defects of ErbB4 null mutant mice were rescued by transgenic expression of an ErbB4 cDNA under a cardiac-specific myosin promoter. This allowed the generation of ErbB4 mutants that develop into adulthood and are fertile. In the second approach, the role of ErbB4 during mammary gland development was specifically addressed by Cre-mediated deletion of both erbB4 alleles within the mammary epithelium. Bellow we discuss the progress made studying these genetic models in understanding the physiological roles of ErbB4 with a focus on the mammary gland and the nervous system.

Fully protected glycosylated zinc (II) phthalocyanine shows high uptake and photodynamic cytotoxicity in MCF-7 cancer cells

Phthalocyanine photosensitizers are effective in anticancer photodynamic therapy (PDT) but suffer from limited solubility, limited cellular uptake and limited selectivity for cancer cells. To improve these characteristics, we synthesized isopropylidene‐protected and partially deprotected tetra β‐glycosylated zinc (II) phthalocyanines and compared their uptake and accumulation kinetics, subcellular localization, in vitro photocytotoxicity and reactive oxygen species generation with those of disulfonated aluminum phthalocyanine. In MCF‐7 cancer cells, one of the compounds, zinc phthalocyanine {4}, demonstrated 10‐fold higher uptake, 5‐fold greater PDT‐induced cellular reactive oxygen species concentration and 2‐fold greater phototoxicity than equimolar (9 μm) disulfonated aluminum phthalocyanine. Thus, isopropylidene‐protected β‐glycosylation of phthalocyanines provides a simple method of improving the efficacy of PDT.