Figure 6: Measurement of proliferation and myogenic differentiation of MDSPCs co-cultured with young WT-MDSPCs.

(a) A schematic diagram of the co-culture system used to evaluate the effect of young functional MDSPCs on dysfunctional MDSPCs isolated from progeroid or old WT mice. Ercc1^−/− MDSPCs were plated in the lower compartment of the transwell system in proliferation media. WT-MDSPCs were seeded onto the upper transwell membrane inserts, at the same density, and in the same media. These co-cultures were placed in the LCI system for 72 h to acquire time-lapsed images to measure proliferation of the MDSPCs. As a control, each plate contained wells of Ercc1^−/− MDSPCs without transwell membrane inserts. (b) Plotted is the average cell number at each time point calculated from the analysis of three independent populations of Ercc1^−/− MDSPCs co-cultured with young WT-MDSPCs (black line), or without (red line) ±s.d. *P<0.001, Mann–Whitney rank sum test. (c) The transwell inserts were removed after 72 h and the proliferation media was switched to differentiation media. After 2–3 days, myogenic differentiation of Ercc1^−/− and old WT-MDSPC was tested by immunostaining the cells for f-MyHC (red). Shown are representative images. The nuclear counterstain is DAPI (blue). Scale bar=100 μm. (d) Quantification of myogenic differentiation of old WT-MDSPCs after growth in media conditioned from young WT-MDSPCs. Young WT-MDSPCs were cultured for 2 days in proliferation media in collagen-coated flasks, then treated with differentiation media for 3 days. The supernatant from these cultures was collected for use as conditioned media. MDSPCs from 21-day-old progeroid Ercc1^−/− mice and 2-yr-old WT mice were grown in the presence of this conditioned media or unconditioned differentiation media to determine the impact on myogenic differentiation as measured by immunoblot detection of f-MyHC. Densitometric quantification of f-MyHC corrected for β-Actin is indicated below each lane.