Induction of osteoprogenitor cell differentiation in rat marrow stroma increases mitochondrial retention of rhodamine 123 in stromal cells.

Bone marrow stromal cells contain colony forming cells with the potential to differentiate into osteoprogenitor (OPC) cells. OPC-stimulation medium, containing dexamethasone, ascorbate, and beta-glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC-stimulation medium on the mitochondrial membrane potential of stromal cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC-stimulation medium showed 2 Rho retention peaks on days 3-4 and 10-11. Between days 5 and 10 there was a drop in Rho retention/cell. OPC-stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 micrograms/ml inhibited the portion of increased Rho retention which was contributed by the OPC-stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 micrograms/ml Rho for 12 days only slightly increased day 12 ALP activity/cell, had no effect on day-21 mineralization and only the high dose, 10.0 micrograms/ml, doubled stromal cell proliferation. Under 24 h incubation Rho concentrations of 1.0 microgram/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal cells. The results indicate that under both culture conditions stromal cell mitochondria undergo cycles of high and low membrane potential states and that the OPC-stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.