A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

Abstract

Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening.

Fig 1. Double fluorescent labeling.

(A) Diagram of cGFP+nmCherry and cmCherry+nGFP–. (B) Phase and fluorescent images for the different labeling combinations, as indicated (scale bar 200 μm) (C, D) Contrast comparison between labels for cytoplasm and nucleus. * p < 0.001 on each day (E) Marking of fiber and nuclei for fusion index measurements in cGFP+nmCherry cells, white outline for myotubes, magenta spots for nuclei inside of fibers, and blue spots for nuclei outside of fibers (scale bar 50 μm).

Fig 2. Immunostaining and live cell imaging of C2C12 fusion.

(A) Representative images of cGFP (green) and nmCherry(red) in parallel with MyHC (red) and DAPI (green) immunostaining, showing differences between matched (magenta) and mismatch (blue) regions. (B,C) Calculated nuclei counts and fusion indices in both methods (scale bar 200μm). * p<0.05.

Fig 3. MYF5 overexpression induces myoblast differentiation.

The effects of Myf5 overexpression on C2C12 cell growth (B) and fusion (A,C) was determined using the double fluorescent cell labeling technique with images shown for day 7 (scale bar- 100μm). *p<0.001.

Fig 4. Activation of WNT signaling inhibits myoblast growth and differentiation.

The effects of the Wnt agonist BIO on C2C12 cell growth in all days and specifically for day 10 (A,D) and fusion (B,C,E) was determined using the double fluorescent cell labeling technique with images shown for day 10 (scale bar- 100μm). * p<0.05.

Fig 5. Exposure to TNFα inhibits myoblast differentiation but not growth.

The effects of TNFα on C2C12 cell growth (A,D) and fusion (B,C,E) was determined using the double fluorescent cell labeling technique (scale bar- 100μm). *p<0.05.

Fig 6. Collagen I coating of scaffolds improves myoblast growth and differentiation.

(A) Scanning emission microscopy of electrospun scaffold coated with collagen I. The effects of Collagen I coating on C2C12 cell growth (C) and fusion (B,D) was determined using the double fluorescent cell labeling technique. (Scale bar-200μm).