An in vitro model to study human NMJ development
Author
Tamás, Michal
Term
4. term
Publication year
2019
Pages
44
Abstract
Sarkopeni er knyttet til degeneration af den neuromuskulære forbindelse (NMJ), men der mangler menneskelige in vitro-modeller til at belyse, hvordan NMJ udvikles. Dette projekt havde til formål at etablere et in vitro-system med primære humane myoblaster for at undersøge effekten af agrin og den gliaafledte neurotrofe faktor (GDNF) på myotubedifferentiering og acetylkolinreceptor (AChR) udtryk, samt at opsætte en heterolog co-kultur af humane myoblaster og rotte-neuroner som et tidligt NMJ-modelsystem. CD56+-myoblaster blev beriget ved magnetisk celle-separation (MACS), og differentiering med tilsætning af agrin eller GDNF blev vurderet ved immunocytokemi (ICC) og kvantitativ PCR (qPCR) af AChR-relaterede gener (CHRND, CHRNG, MUSK). Derudover blev neuritvækst analyseret i co-kultur og neuralt medium. Under de testede betingelser fremmede agrin hverken myotubedifferentiering eller AChR-klyngedannelse. GDNF gav anledning til let større, multinukleære myotuber og opregulerede AChR-relaterede transkripter. Neuroner i co-kultur og neuralt medium udviklede længere og flere neuritter. Modne AChR-klynger, som indikerer fuldt dannede NMJ, blev ikke observeret, hvilket tyder på, at yderligere faktorer eller længere kulturforløb kan være nødvendige. Arbejdet repræsenterer et første skridt mod en menneskerelevant in vitro-model for NMJ-udvikling og peger på GDNF som en modulator af postsynaptisk differentiering, mens der er behov for yderligere optimering for at opnå modne NMJ-strukturer.
Sarcopenia is linked to degeneration of the neuromuscular junction (NMJ), yet human in vitro models are needed to clarify how NMJs develop. This project aimed to establish an in vitro system with primary human myoblasts to test the effects of agrin and glial cell line–derived neurotrophic factor (GDNF) on myotube differentiation and acetylcholine receptor (AChR) expression, and to set up a heterologous co-culture of human myoblasts with rat neurons as an early NMJ model. CD56+ myoblasts were enriched by magnetic cell separation (MACS), and differentiation with added agrin or GDNF was evaluated by immunocytochemistry (ICC) and quantitative PCR (qPCR) for AChR-related genes (CHRND, CHRNG, MUSK). Neurite outgrowth was also assessed in co-culture and neural medium. Under the tested conditions, agrin did not promote myotube differentiation or AChR clustering. GDNF produced slightly larger, multinucleated myotubes and upregulated AChR-related transcripts. Neurons in co-culture and neural medium extended longer and more neurites. Mature AChR clusters indicative of fully formed NMJs were not detected, suggesting that additional factors or longer culture durations may be required. This work provides initial steps toward a human-relevant in vitro model of NMJ development and highlights GDNF as a modulator of postsynaptic differentiation while underscoring the need for further optimization to achieve mature NMJ formation.
[This summary has been generated with the help of AI directly from the project (PDF)]
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