Embryonic motoneuron-skeletal muscle co-culture in a defined system

A system for studying mechanisms of neuromuscular junction development and maintenance

“…However, we were particularly interested in characterizing the differentiation of muscle fibers in a neuronal context, replicating in vivo conditions. Much attention has been given to the development and improvement of the neuronal compartment with the emergence of embryonic stem cells (Chipman et al, 2014;Das et al, 2007;Soundararajan et al, 2007) and induced pluripotent stem cells (Bohl et al, 2015;Burkhardt et al, 2013;Egawa et al, 2012); however, the muscle counterpart has been underexploited and its potential underestimated. Previous studies failed to produce convincing data on muscle differentiation either because muscle cell lines, such as C2C12, incapable of producing highly differentiated myofibers, were used or because the muscle compartment was overlooked (Arnold et al, 2012;Chipman et al, 2014;Das et al, 2009Das et al, , 2010Umbach et al, 2012).…”

“…In several previous protocols that described NMJ formation, the researchers have presented NMJs as random colocalization points between the neuron and the myofiber with dotty and/or unstructured AChR clusters (Das et al, 2007;Southam et al, 2013), usually formed before any innervation. However, in vivo, neuron-muscle fortuitous colocalization alone is not a satisfactory feature (Thomson et al, 2012).…”

“…To date, co-culture methods established from various species have been described, including mouse (Morimoto et al, 2013;Zahavi et al, 2015), rat (Das et al, 2010;Southam et al, 2013), Xenopus (Lu et al, 1996;Peng et al, 2003) and chick (Frank and Fischbach, 1979), and also heterologous co-cultures built from motor neuron and muscle cells obtained from different species, such as rat-human , mouse-human (Son et al, 2011) and mouse-chick (Soundararajan et al, 2007). However, these co-culture methods resulted in the formation of immature myofibers (thin muscle fiber, with centrally localized nuclei and no transversal triads) with immature sarcomeric structures (Das et al, 2007(Das et al, , 2009Southam et al, 2013). Moreover, previous models did not take advantage of their co-culture system to analyze other post-synaptic structures such as the formation of muscle-specific tyrosine kinase (MuSK) and Rapsyn (also known as Rapsn) clusters which are formed as agrin-induced signaling sparks off and which are essential to the formation of acetylcholine receptor (AChR) clusters.…”

Fetal bovine serum (FBS): Past – present – future

“…Also, quality and yield of cell culture-based biotechnology products, diagnostics and vaccines may have been affected (Gstraunthaler et al, 2014). As a very visible result of this turmoil in the serum market, the jan et al., 2006, 2011), hippocampal neurons (Varghese et al, 2009), motoneurons (MNs) (Das et al, 2003(Das et al, , 2007aGuo et al, 2010), sensory neurons (Guo et al, 2013;Rumsey et al, 2010), muscle (Das et al, 2006(Das et al, , 2007bVarghese et al, 2009), neuromuscular junction formation Guo et al, 2010;Smith et al, 2013), and a multi-organ system with functional readout (Oleaga et al, 2016).…”

Toward in vitro models of brain structure and function

“…Frampton et al (4) used alginate with rat astroglioma cells, astrocytes, microglia, and neurons, but did not attempt to mimic the layered architecture of the cerebral cortical tissue or demonstrate electrical connections that will be critical to a more complete understanding of brain function. Functional culture has been shown with neuromuscular junction systems for rat (8) and human (9), but without the accompanying 3D architecture.…”