Gaia Carbone
Characterization of Cellular Differentiation in an in vitro model of Neuromuscular Junction using a Co-Culture system: A Preliminary Study of a Drug Testing Platform
Autori
- GAIA CARBONE (UNIVERSITÀ DEGLI STUDI DI BARI – DOTTORANDA)
- VITTORIA CANFORA (UNIVERSITÀ DEGLI STUDI DI BARI – ASSEGNISTA DI RICERCA)
- SABATA PIERNO (UNIVERSITÀ DEGLI STUDI DI BARI – PROFESSORE ASSOCIATO)
- GIULIA MARIA CAMERINO (UNIVERSITÀ DEGLI STUDI DI BARI – PROFESSORE ASSOCIATO)
Presentatore
GAIA CARBONE
Modalità
Poster Session
Abstract
“Neuromuscular junction (NMJ) alteration is involved in a variety of diseases, among them, the Amyotrophic Lateral Sclerosis. In vitro models of the NMJ have proven to be invaluable platforms for testing new drugs. In this preliminary study, our goal was to establish a simple and adaptable in vitro platform for drug investigations. We focused on the cellular differentiation preceding the formation of the NMJ using a co-culture system derived from immortalized muscle (C2C12) and neuronal (NCS-34) cells, as previously described (Taro Inoue et al. 2021).
We assessed the expression of genes associated with neuronal (Hb9 and Map2) and muscular (Mhc1, MyoD, MyoG, and AChRE) differentiation in the co-culture at 0, 7, and 9 days of differentiation (0D, 7D, 9D) by RT-PCR. Additionally, differentiation was examined at 9D, through immunofluorescence analysis using alpha-bungarotoxin and antibodies targeting peripherin and laminin.
The expression of MyoG and Map2 increased from 0D to 9D, while Mhc1 and MyoD expression was only detectable at 9D. However, the expression of mature NMJ markers (Hb9 and AChRE) was not detectable at 9D. Immunofluorescence with laminin revealed the initiation of differentiation in both cell types, including myocyte formation. Peripherin exhibited the development of elongated neurites in NSC-34 cells, while alpha-bungarotoxin was not detectable.
After 9 days of co-culture, both C2C12 and NSC-34 cells began their differentiation process, but the synaptic junction still remained immature. The study of cell differentiation before the NMJ has formed can help in understanding mechanisms underlying the impairment in neuromuscular diseases.”