Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Our research involves the study of healthy and diseased skeletal muscle with special emphasis on the satellite cells. One of the fundamental aspects we are exploring is the identification of transcriptional enhancers that coordinate cell specific gene expression. This protocol permits us to conduct rapid tests for determining specificity.

In the field of skeletal muscle research, single cell and single nucleus RNA sequencing approaches are frequently used to study different cell populations in homeostasis, regeneration, and disease. Also, satellite cells could be isolated by fluorescence activated cell sorting, or FACS, and cultured for hypothesis testing and sequencing result validation. Studying satellite cells in vitro poses a significant challenge.

Recent studies highlighted the role of satellite cells niche in its physiology. While it’s common to culture FACS isolated satellite cells, replicating the satellite cells niche in vitro is yet to be achieved. Our protocol provides an efficient and practical way of culturing whole muscle tissue, including non myogenic cells, such as endothelial cells and fibro edoprogenetic progenitors.

We showed that quiescent-like cells appear within one week of culture. This protocol could also be used to study the influence that other cells have on reestablishing this quiescent-like pool.