Generating intrafusal skeletal muscle fibres in vitro: Current state of the art and future challenges

Abstract

Intrafusal fibres are a specialised cell population in skeletal muscle, found within the muscle spindle. These fibres have a mechano-sensory capacity, forming part of the monosynaptic stretch-reflex arc, a key component responsible for proprioceptive function. Impairment of proprioception and associated dysfunction of the muscle spindle is linked with many neuromuscular diseases. Research to-date has largely been undertaken in vivo or using ex vivo preparations. These studies have provided a foundation for our understanding of muscle spindle physiology, however, the cellular and molecular mechanisms which underpin physiological changes are yet to be fully elucidated. Therefrom, the use of in vitro models has been proposed, whereby intrafusal fibres can be generated de novo. Although there has been progress, it is predominantly a developing and evolving area of research. This narrative review presents the current state of art in this area and proposes the direction of future work, with the aim of providing novel pre-clinical and clinical applications.

Neuregulin 1 Drives Morphological and Phenotypical Changes in C2C12 Myotubes: Towards De Novo Formation of Intrafusal Fibres In Vitro

“…This indicates the probable biological significance of Myh4 and Myh3 fold changes seen in Figure 4A , as they represent the greatest proportional expression in both control and Nrg-1 treated myotubes. This MyHC RT-qPCR data demonstrates a unique contractile phenotype in the treated population, however we see decreased expression in Myh isoforms associated with intrafusal fibres in vivo and in vitro ( Barrett et al, 2020 ).…”

“…In turn, 3D skeletal muscle cultures permit more mature molecular, structural and functional differentiation, better representing native adult muscle (Rao et al, 2018;Capel et al, 2019;Wang et al, 2019;Zhuang et al, 2020) and may facilitate identification of retained immature or specialised intrafusal specific phenotypes using C2C12 cells. Other novel future approaches are discussed in detail in a recent review paper by our group (Barrett et al, 2020). Without a definitive intrafusal phenotype in vitro, we cannot speculate on the origins of intrafusal fibres.…”

“…There is enough promise from the results presented here to encourage future research toward biomimetic tissue engineering approaches, in the hope of producing mature, Nrg-1 treated myotubes, characteristic of native, in vivo intrafusal skeletal muscle. These future models could provide platforms for developmental or disease state studies, pre-clinical screening, or clinical applications, including regeneration or replacement of diseased or dysfunctional tissue ( Barrett et al, 2020 ; Kröger and Watkins, 2021 ).…”

“…In vitro skeletal muscle research has gained momentum over the past 10 years, however there are still relatively few publications tackling cell and tissue engineering approaches to generating de novo intrafusal muscle fibres in vitro ( Barrett et al, 2020 ) and they are not without some inconsistencies, described previously. This study presents an objective, novel method for assignment of intrafusal bag morphology and the most in-depth characterisation of myotubes following Nrg-1 treatment to-date.…”

Emerging biogenesis technologies of extracellular vesicles for tissue regenerative therapeutics

“…Among the EVs isolation techniques, the ultracentrifuge-and-filtration is the most widely used that can collect EVs with relatively confined size, but it needs large sample volume and is time-consuming. 8 – 10 Microfluidics method requires low sample volume and cost, but the isolated EVs amount per time is limited. On the other hand, the asymmetric flow field-flow fractionation technique can collect EVs in a gentle, rapid (<1 h) and highly reproducible way, but the particle loss and variation in the size due to sticking to the barrier membrane should be improved.…”