Research Paper Volume 4, Issue 8 pp 553—566

Figure 3. Osteopontin inhibits regenerative responses of muscle stem cells in co-cultures with CD11b+ cells.

(A) Schematic representation of transwell culture assay. Myofiber explants (satellite cells associated with myofibers) were obtained from 3 DPI young and old TA, were plated on ECM coated lower chamber of transwell in their respective 5% serum whereas MACS isolated CD11b+ macrophages from the same injured muscles of young and old mice were isochronically or hetrochronically plated on 0.3 μm PE membrane transwell insert. At the time of plating, these co-cultures were treated (or not) with rOPN or nOPN antibody, and after 20 hours, co-cultures were pulsed with BrdU for 4 hours and subsequently, fixed and co-immunostained for BrdU and Desmin. (B) Representative pictures of YYY (Young myofiber in Young mouse serum with Young CD11b+ macrophages), YYY + rOPN (recombinant OPN), OOO (Old myofiber in old mouse serum with Old CD11b+ macrophages) and OOO + nOPN (neutralization of OPN) are shown. (C) Quantification of the myogenicity in the above-described transwell co-cultures. Bar graphs represent the mean and standard deviation in percent of BrdU+/Desmin+ proliferating myogenic cells; n=3, ±S.E.M. Young macrophages or neutralization of OPN increased the clonogenic / myogenic capacity of old satellite cells that associate with old myofibers and cultured in old serum.