A 3D cell culture system for bioengineering human neuromuscular junctions to model ALS

Abstract

The signals that coordinate and control movement in vertebrates are transmitted from motoneurons (MNs) to their target muscle cells at neuromuscular junctions (NMJs). Human NMJs display unique structural and physiological features, which make them vulnerable to pathological processes. NMJs are an early target in the pathology of motoneuron diseases (MND). Synaptic dysfunction and synapse elimination precede MN loss suggesting that the NMJ is the starting point of the pathophysiological cascade leading to MN death. Therefore, the study of human MNs in health and disease requires cell culture systems that enable the connection to their target muscle cells for NMJ formation. Here, we present a human neuromuscular co-culture system consisting of induced pluripotent stem cell (iPSC)-derived MNs and 3D skeletal muscle tissue derived from myoblasts. We used self-microfabricated silicone dishes combined with Velcro hooks to support the formation of 3D muscle tissue in a defined extracellular matrix, which enhances NMJ function and maturity. Using a combination of immunohistochemistry, calcium imaging, and pharmacological stimulations, we characterized and confirmed the function of the 3D muscle tissue and the 3D neuromuscular co-cultures. Finally, we applied this system as an in vitro model to study the pathophysiology of Amyotrophic Lateral Sclerosis (ALS) and found a decrease in neuromuscular coupling and muscle contraction in co-cultures with MNs harboring ALS-linked SOD1 mutation. In summary, the human 3D neuromuscular cell culture system presented here recapitulates aspects of human physiology in a controlled in vitro setting and is suitable for modeling of MND.

Neuronal Circuit Dysfunction in Amyotrophic Lateral Sclerosis

“…Equally, human in-vitro models have been able to recapitulate NMJ dysfunctions in ALS models [157][158][159][160][161] over a wide range of genotypes, demonstrating that NMJ degeneration is a consistent finding in ALS models. For instance, SOD1 +/G85R and PFN1 +/G118V human neuromuscular organoids lead to a reduced innervation of NMJs [160].…”

Recent Research Trends in Neuroinflammatory and Neurodegenerative Disorders

“…hiPSC-derived neuromuscular organoids provide a reliable model to study the initial cellular pathologies, which is not possible in patients as ALS is diagnosed quite late [ 249 ]. Three-dimensional microfluidic NMJ models have been used to gain insight into the morphological and functional readouts at the NMJ contributing to the ALS pathology [ 250 ].…”

“…hiPSC-derived neuromuscular organoids provide a reliable model to study the initial cellular pathologies, which is not possible in patients as ALS is diagnosed quite late [249]. Three-dimensional microfluidic NMJ models have been used to gain insight into the morphological and functional readouts at the NMJ contributing to the ALS pathology [250]. iPSC-derived mast cells are pivotal in neuroinflammatory responses, generated through protocols recapitulating the in vivo developmental stages by controlled exposure to growth factors, are crucial in conditions like migraine and multiple sclerosis, where mast cell activation exacerbates CNS inflammation, potentially leading to BBB disruption and immune cell recruitment [251].…”

Advancements in 2D and 3D In Vitro Models for Studying Neuromuscular Diseases

“…Familial ALS is associated with pathogenic variants in SOD1. Pathogenic variants in C9orf72, TDP-43, and FUS are associated with familial and sporadic ALS [40,61,67,89]. Massih et al developed an in vitro model that generates NMJs via a co-culture system consisting of iPSC-derived MNs with SOD1 pathogenic variants and 3D skeletal muscle tissue to study NMJ physiology and dysfunction [61].…”

“…Pathogenic variants in C9orf72, TDP-43, and FUS are associated with familial and sporadic ALS [40,61,67,89]. Massih et al developed an in vitro model that generates NMJs via a co-culture system consisting of iPSC-derived MNs with SOD1 pathogenic variants and 3D skeletal muscle tissue to study NMJ physiology and dysfunction [61]. This model exhibits features characteristic of ALS, such as decreased muscle contraction and pathological traits.…”