Simplified in vitro engineering of functional mammalian neuromuscular junctions between embryonic rat motor neurons and immortalised human skeletal muscle cells

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular (NM) pathologies and development of novel therapies for diseases associated with NM dysfunction and deterioration. Several in vivo animal models manifest phenotypes observed in NM diseases. Unfortunately, in vivo NMJ research with animal models present many challenges due to inaccurate reproduction of human disease. For example, the most widely used animal model for Duchenne muscular dystrophy, the mdx mouse, is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle. However, this in vivo model is not useful for clinical trials due to the very mild phenotype expressed. Therefore, in vitro models were established, yet limitations exist. For example, inclusion of serum influences translation of animal data into human trials, inclusion of complex neurotrophic/growth factors can interfere with drug discovery, the initiation of skeletal muscle (SkM) contractions requiring electric pulse or chemical stimuli, and time consuming culture methods to induce spontaneous SkM contractions. Therefore, the aim of this thesis was to establish and characterise a simplified co-culture system that allows in vitro research of functional NMJs, representative of in vivo conditions. Immortalised human SkM stem cells were co-cultured with motor neurons (MNs) from rat embryo spinal cord explants, using for the first time a culture media formulation free from serum and neurotrophic or growth factors. This co-culture resulted in NMJ formation and contractile SkM cells. The de novo formation of NMJs was validated via characterisation of pre- and post-synaptic structures of the junctional apparatus. Interactions between the specialised membranes of presynaptic MN terminals with postsynaptic motor end plates (MEPs) located on SkM cells, along with supporting neuroglia, permitted chemical transmission of acetylcholine from MNs across structural bridges to bind with receptors on the MEPs. These interactions were associated with contractile activity and advanced differentiation of innervated SkM fibres. Functionality of NMJs was verified through the application of known agonists and antagonists to the co-culture system and confirmed that the contractile activity observed in the innervated SkM fibres were driven via NMJs. An ELISA-based microarray identified the presence of trophic factors required for MN, SkM, and NMJ development. Ultimately, engineering of this novel in vitro NMJ system represents an accessible platform to investigate NMJ formation and function, as well as providing a breakthrough assay via the system’s ability to respond to drug interventions through measurable output, initiate spontaneous SkM cell contractions, and induce advanced differentiation of SkM Cells. Therefore, this novel system provides a tool to screen pharmacological or genetic therapies for diseased linked with SkM, MNs, and NMJs

Cross-talk between motor neurons and myotubes via endogenously secreted neural and muscular growth factors.

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular patho-physiology and development of novel therapies for diseases associated with NM dysfunction. In vivo, the micro-environment surrounding the NMJ has a significant impact on NMJ formation and maintenance via neurotrophic and differentiation factors that are secreted as a result of cross-talk between muscle fibers and motor neurons. Recently we showed the formation of functional NMJs in vitro in a co-culture of immortalized human myoblasts and motor neurons from rat-embryo spinal-cord explants, using a culture medium free from serum and neurotrophic or growth factors. The aim of this study was to assess how functional NMJs were established in this co-culture devoid of exogenous neural growth factors. To investigate this, an ELISA-based microarray was used to compare the composition of soluble endogenously secreted growth factors in this co-culture with an a-neural muscle culture. The levels of seven neurotrophic factors brain-derived neurotrophic factor (BDNF), glial-cell-line-derived neurotrophic factor (GDNF), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-1 (IGF-1), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), and vascular endothelial growth factor (VEGF) were higher (p < 0.05) in the supernatant of NMJ culture compared to those in the supernatant of the a-neural muscle culture. This indicates that the cross-talk between muscle and motor neurons promotes the secretion of soluble growth factors contributing to the local microenvironment thereby providing a favourable regenerative niche for NMJs formation and maturation

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction.

BACKGROUND:In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. OBJECTIVE:to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. METHODS:an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. RESULTS:blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. CONCLUSION:a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions

A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.

Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and βIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction

Background: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs. Objective: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function. Methods: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists. Results: blocking acetylcholine receptors with &alpha;-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional. Conclusion: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions

Control strategies for Listeria monocytogenes in ready-to-eat foods and on food contact surfaces

Doctor of PhilosophyFood ScienceDaniel Y.C. FungJames L. MarsdenThe ubiquitous nature and continued presence in food processing environments makes Listeria monocytogenes a significant threat in ready-to-eat (RTE) food products. This study was performed in two phases; Phase 1 studied lauric arginate (LAE) as an antimicrobial on food contact surfaces and shredded mozzarella cheese, and use of glucose oxidase (GOX), sodium lactate (SL), and acidified calcium sulfate (ACS) as preservatives in mozzarella cheese; Phase 2 evaluated efficacy of Photohydroionization (PHI) technology to control L. monocytogenes on food contact surfaces, sliced American cheese, and ready-to-eat turkey. Stainless steel coupons, mozzarella cheese, American cheese, and turkey were surface inoculated with a three- or five-strain cocktail of L. monocytogenes. Coupons were treated with 100 and 200 ppm solution of lauric arginate for 5 and 15 min. Mozzarella cheese was treated with different combinations of treatments comprising LAE, GOX, SL, ACS, dextrose, and anticaking agents (free flow 1031 and cellulose). Results indicated up to 2.5 log CFU/coupon reductions and it was concluded that LAE was effective in controlling low levels of contamination of L. monocytogenes on food contact surfaces. In mozzarella cheese, results indicated that lauric arginate provided no additional antimicrobial effect (P > 0.05) as compared to GOX + dextrose. The antimicrobial blends with GOX, SL, and ACS were different (P 0.05) in their effect in controlling bacterial populations. Results from treatment with PHI unit showed significant (P 0.05) TBAR values. These studies suggest that LAE and GOX as antimicrobials and PHI treatment can be used as intervention strategies in an integrated process to ensure safe production of food. Further research is needed to evaluate applicability of SL and ACS in mozzarella cheese

Validating the efficacy of commercial foaming cleaner and sanitizer for controlling Listeria innocua (surrogate for Listeria monocytogenes) in drains and potential translocation from the drain to the food contact surfaces

Master of ScienceFood Science InstituteDaniel Y.C. FungJames L. MarsdenListeria monocytogenes is known to be an environmental contaminant in food processing facilities. Floor drains in processing environments harbor Listeria spp. due to continuous presence of humidity and organic substrates. The cleaning and washing activities undertaken may translocate the bacterial cells from the drain to the surrounding environment, thus contaminating food products being produced. This study validates the effectiveness of Johnson Diversey ‘Eliminex’ Foaming Drain Cleaner and Johnson Diversey ‘Final Step’ 512 sanitizer for inhibition of Listeria monocytogenes in drain surfaces and evaluates the potential for translocation of L. monocytogenes from drains to food contact surfaces in the surrounding environment using Listeria innocua as a surrogate. A 7x 7 x 8 feet flexi glass chamber was built in which a 10 inch diameter drain mounted on an aluminum cabinet was placed. The drain was inoculated with the surrogate organism, L. innocua, at specific time intervals and then treated with the given chemicals. Sponge samples were taken and bacterial populations were recovered on Tryptic Soy Agar (TSA), Modified Oxford Medium (MOX) and Thin Agar Layer MOX (TALMOX). Stainless steel coupons (6.4 x 1.9 x 0.1 cm) were hung at 3 different heights 1, 3 and 5 feet inside the chamber and cell translocation from the drain on to the stainless steel coupons was studied. Reductions up to 4 Log CFU/area or ml were seen at the drain surface, drain crate, drain pipe and wash water for both free cells and cells entrapped in biofilms Treatment had a significant effect (p<0.05) on the reduction of bacterial cells. The wash water showed the greatest reduction from 8 Log CFU/ml to est. 0.23 Log CFU/ml. The given cleaner and sanitizer were found to be effective for reducing Listeria spp. on drain surfaces. Results for the second part indicated translocation at all three heights with percentage translocation ranging between 2-17%. Significantly higher translocation (p<0.05) was seen at 1 foot, followed by 3 feet and 5 feet indicating the closer the height to the drain, the greater the number of bacterial cells that are able to transfer from the drain to the surrounding environment