Anita F. Quigley

A conducting-polymer platform with biodegradable fibers for stimulation and guidance of axonal growth

A biosynthetic platform composed of a conducting polypyrrole sheet embedded with unidirectional biodegradable polymer fibers is described (see image; scale bar = 50 µm). Such hybrid systems can promote rapid directional nerve growth for neuro-regenerative scaffolds and act as interfaces between the electronic circuitry of medical bionic devices and the nervous system.

In Vivo and in Vitro Correction of the mdx Dystrophin Gene Nonsense Mutation by Short-Fragment Homologous Replacement

Targeted genetic correction of mutations in cells is a potential strategy for treating human conditions that involve nonsense, missense, and transcriptional splice junction mutations. One method of targeted gene repair, single-stranded short-fragment homologous replacement (ssSFHR), has been successful in repairing the common deltaF508 3-bp microdeletion at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in 1% of airway epithelial cells in culture. This study investigates in vitro and in vivo application of a double-stranded method variant of SFHR gene repair to the mdx mouse model of Duchenne muscular dystrophy (DMD). A 603-bp wild-type PCR product was used to repair the exon 23 C-to-T mdx nonsense transition at the Xp21.1 dys locus in cultured myoblasts and in tibialis anterior (TA) from male mdx mice. Multiple transfection and variation of lipofection reagent both improved in vitro SFHR efficiency, with successful conversion of mdx to wild-type nucleotide at the dys locus achieved in 15 to 20% of cultured loci and in 0.0005 to 0.1% of TA. The genetic correction of mdx myoblasts was shown to persist for up to 28 days in culture and for at least 3 weeks in TA. While a high frequency of in vitro gene repair was observed, the lipofection used here appeared to have adverse effects on subsequent cell viability and corrected cells did not express dystrophin transcript. With further improvements to in vitro and in vivo gene repair efficiencies, SFHR may find some application in DMD and other genetic neuromuscular disorders in humans.

Mitochondrial respiratory chain activity in idiopathic dilated cardiomyopathy

BACKGROUND Cardiomyopathy is well recognized in mitochondrial diseases in which it has been associated with defects of mitochondrial function, including cytochrome-c oxidase (COX) deficiencies. This study explores the respiratory chain activity, particularly of COX, in patients with cardiomyopathy to determine whether a relationship exists between respiratory enzyme activity and cardiac function. METHODS AND RESULTS Myocardial specimens from the left ventricular wall of explanted hearts were obtained from subjects with ischemic (n = 6) or nonischemic dilated (n = 8) cardiomyopathy. Assays for citrate synthase (CS) and complexes II/III and IV activity were performed on cardiac mitochondria and homogenate. Enzyme activities were normalized to CS activity and compared with control activities (n = 10). A significant reduction in COX and/or CS activity was identified in mitochondrial preparations from the transplant group and correlated significantly with ejection fraction (P < .05), although this does not prove a causal relationship. Significantly reduced CS activity in homogenate was identified, suggesting decreased mitochondrial volume in addition to decreased COX activity. Measurements in cardiac homogenates failed to show a significant reduction in COX activity (P > .05) in the transplant group, suggesting that the use of prefrozen tissue homogenates may underestimate existing mitochondrial respiratory defects in cardiac tissue. CONCLUSIONS Mitochondrial function is altered at a number of levels in end-stage cardiomyopathy. Defective COX activity resulting in deficient adenosine triphosphate generation may contribute to impaired ventricular function in heart failure. Agents capable of improving mitochondrial function may find an adjuvant role in the treatment of cardiac failure.

Detection of MELAS A3243G point mutation in muscle, blood and hair follicles

Polymerase chain reaction (PCR) based methods for the diagnosis and screening of the mitochondrial disorders have been well established. A number of tissues are routinely used. In this study, we compared the detection rate for MELAS A3243G point mutation in muscle, blood and hair follicles. Ten subjects were studied; mean age was 47 years, (SD 16, range 23-73). All ten subjects had the MELAS A3243G point mutation detected in muscle and hair follicles, but only five had the abnormality in blood samples. The rate of detection of the point mutation in blood samples was age dependent. MtDNA analysis on hair follicles is as sensitive as muscle in detecting this mutation. Analysis using blood samples is not as sensitive, particularly in older subjects. The absence of the mutation in blood samples suggests that there is a preferential selection process for normal (wild type) mtDNA over time. This may be related to the rate of cell division and energy requirements of each tissue.

Chondrogenesis of Infrapatellar Fat Pad Derived Adipose Stem Cells in 3D Printed Chitosan Scaffold

Infrapatellar fat pad adipose stem cells (IPFP-ASCs) have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a ‘cap’ on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

Electrical Stimulation of Myoblast Proliferation and Differentiation on Aligned Nanostructured Conductive Polymer Platforms

In this study, nanostructured conductive platforms synthesized from aligned multiwalled carbon nanotubes and polypyrrole are investigated as myo-regenerative scaffolds. Myotube formation follows a linear path on the platforms coinciding with extent of nanotopography. In addition, electrical stimulation enhances myo-nuclear number and differentiation. These studies demonstrate that conductive polymer platforms can be used to influence muscle cell behaviour through nanostructure and electrical stimulation.

Mitochondrial DNA in Stroke and Migraine with Aura

Patients presenting with thrombotic stroke of unexplained etiology and or migraine with aura were screened for mitochondrial (mt) DNA mutations associated with cytopathies given that both migraine and stroke-like episodes are recognised with certain mt DNA mutations. Mutations usually associated with either mitochondrial encephalopathy, lactic acidosis and stroke-like episode, myoclonic epilepsy with ragged red fibres, or those strongly linked to Leber’s hereditary optic neuropathy (LHON) were not detected in patients or controls. However, increased levels of two of the secondary LHON mutations were found. The T→C mutation at nucleotide 4216 was more common than expected in patients aged 35 years or less, as was the 13708 G→A mutation in young stroke patients. This data lends support to the possibility that an accumulation of minor mt DNA mutations may contribute to the pathoaetiology of stroke and migraine with aura in some young patients.

Targeted gene correction in the mdx mouse using short DNA fragments: towards application with bone marrow-derived cells for autologous remodeling of dystrophic muscle

In muscle, mutant genes can be targeted and corrected directly by intramuscular (i.m.) injection of corrective DNA, or by ex vivo delivery of DNA to myogenic cells, followed by cell transplantation. Short fragment homologous replacement (SFHR) has been used to repair the exon 23 nonsense transition at the Xp21.1 dys locus in cultured cells and also, directly in tibialis anterior from male mdx mice. Whilst mdx dys locus correction can be achieved in up to 20% of cells in culture, much lower efficiency is evident by i.m. injection. The major consideration for application of targeted gene correction to muscle is delivery throughout relevant tissues. Systemically injected bone marrow (BM)-derived cells from wt C57BL/10 ScSn mice are known to remodel mdx muscle when injected into the systemic route. Provided that non muscle-derived cell types most capable of muscle remodeling activity can be more specifically identified, isolated and expanded, cell therapy seems presently the most favorable vehicle by which to deliver gene correction throughout muscle tissues. Using wt bone marrow as a model, this study investigates systemic application of bone marrow-derived cells as potential vehicles to deliver corrected (ie wt) dys locus to dystrophic muscle. Intravenous (i.v.) and intraperitoneal (i.p.) injections of wt BM were given to lethally and sub-lethally irradiated mdx mice. Despite both i.v. and surviving i.p. groups containing wt dys loci in 100% and less than 1% of peripheral blood nuclei, respectively, both groups displayed equivalent levels of wt dys transcript in muscle RNA. These results suggest that the muscle remodeling activity observed in systemically injected BM cells is not likely to be found in the hemopoietic fraction.

Diagnostic screening of mitochondrial DNA mutations in Australian adults 1990-2001

Abstract

Electrical stimulation using conductive polymer polypyrrole promotes differentiation of human neural stem cells: a biocompatible platform for translational neural tissue engineering.

Conductive polymers (CPs) are organic materials that hold great promise for biomedicine. Potential applications include in vitro or implantable electrodes for excitable cell recording and stimulation and conductive scaffolds for cell support and tissue engineering. In this study, we demonstrate the utility of electroactive CP polypyrrole (PPy) containing the anionic dopant dodecylbenzenesulfonate (DBS) to differentiate novel clinically relevant human neural stem cells (hNSCs). Electrical stimulation of PPy(DBS) induced hNSCs to predominantly β-III Tubulin (Tuj1) expressing neurons, with lower induction of glial fibrillary acidic protein (GFAP) expressing glial cells. In addition, stimulated cultures comprised nodes or clusters of neurons with longer neurites and greater branching than unstimulated cultures. Cell clusters showed a similar spatial distribution to regions of higher conductivity on the film surface. Our findings support the use of electrical stimulation to promote neuronal induction and the biocompatibility of PPy(DBS) with hNSCs and opens up the possibility of identifying novel mechanisms of fate determination of differentiating human stem cells for advanced in vitro modeling, translational drug discovery, and regenerative medicine.