FIGURE 2:

Constricted migration of myoblasts delays differentiation. (A) (Left) C2C12 cells are allowed to migrate through 3- or 8-μm transwell pores over the course of 24 h in growth media. Migrated cells (on the bottom [Bot.] of the transwell pore filter) and nonmigrated cells (on the top [Top]) are then collected separately and plated at high density in growth media. After 3 h of adhesion, cells are switched to differentiation media and cultured for 0–6 d. (Right) Representative images of C2C12 myoblasts 0–6 d after migration through 3- or 8-μm pores. Differentiation is indicated by immunofluorescent staining for skeletal myosin heavy chain (Skel.MyoII). Scale bar = 20 μm. (B) (i) Compared with either nonmigrated or 8 μm pore–migrated cells, cells undergoing 3-μm pore migration show significantly reduced differentiation over days 2–3, resulting in an ∼3-d delay in differentiation. N = 3–15 samples. (ii) However, 3 μm pore–migrated cells also show significantly reduced cell density throughout the differentiation time-course. (C) (i) Even when 8 μm pore–migrated cells are plated at low initial density to match the reduced cell density of 3 μm pore–migrated cells, the day-3 differentiation defect persists. N = 3 samples. (ii) When 8 μm pore–migrated cells are plated at low initial density, the 3- and 8-μm populations maintain similar densities throughout the differentiation time course. (D) (i) Differentiation index at day 3 is a linear function of cell density at either day 0 or day 1 (data from Supplemental Figure S2, B and C). The differentiation index for 3 μm pore–migrated cells falls below the line, indicating less differentiation than expected based on cell density. x_i denotes the density expected for differentiation, and d_i denotes the excess density. (ii) The excess density for the expected population-level differentiation decays over days as cells recover from migrating through 3-μm pores. (E) (Top) After 24 h of migration, cells are collected from the bottom of the transwell membrane, plated in growth media for 4–5 d to allow proliferation, and then reseeded on top of a new transwell membrane. This process is repeated twice. Following the third migration, cells are allowed to proliferate to confluence and then differentiated for 4 d. (Bottom) Representative images of cells either cultured in 2D or subjected to three times–repeated migration through 3- or 8-μm pores. Myoblasts exhibit very little differentiation, as indicated by Skel.MyoII, after repeated migration through 3-μm pores. Scalebar = 20 μm. (F) Quantification of differentiation index normalized to 2D: cells undergoing constricted migration have a differentiation defect that persists following 4–5 d of recovery growth and 4 d of differentiation. N = 6 samples. * indicates significant (p < 0.05) difference from 8-μm migration. (G) Cells were grown for 8 rather than 4–5 d before 4-d differentiation. This extended recovery time between serial migration assays eliminates the differentiation defect in 3-μm pore-migrated cells. N = 3 samples.