Fig. 2. Differentiation potential of AGM cells. (A-C) Osteogenic cell differentiation. After 10 days in osteogenic permissive medium, cell colonies stained positive for ALP activity (A), and cultures expressed the osteoblastic markers osteopontin and osteocalcin (B, lane 2). Neither marker was expressed in freshly isolated AGM cells (B, lane1). Differentiated cultures also showed an upregulation of the osteogenic marker Cbfa1 (B, lane 2). At day 21, mineralization of the cultures was observed with Alizarin Red (C). (D) Adipogenic differentiation and colony formation. After stimulation with adipogenic-inductive medium for one week, Pparg was upregulated (B, lane 2) when compared with the expression of this marker in freshly isolated AGM cells (B, lane 1). (E-H) Chondrogenic differentiation. After 21 days in chondrogenic medium, cells formed several cartilaginous foci (E) with an extracellular matrix rich in proteoglycans, as detected by Toluidine Blue staining (F). Foci were also strongly positive for collagen type II, as shown by the brown DAB staining (G). A secondary antibody control is shown in H.