PRELIMINARY CHARACTERIZATION OF PORCINE BONE-MARROW STROMAL CELLS - SKELETOGENIC POTENTIAL, COLONY-FORMING ACTIVITY, AND RESPONSE TO DEXAMETHASONE, TRANSFORMING GROWTH-FACTOR-BETA, AND BASIC FIBROBLAST GROWTH-FACTOR

Neonatal pig bone marrow stromal cells (PBMSC) were tested in vivo and in vitro to establish their use as a large-animal model for the study of skeletogenesis. When implanted in diffusion chambers in athymic mi ce for 6-8 weeks, both freshly isolated pig bone marrow and passage 2 PBMSC formed partially mineralized cartilage, bone-like material, and fibrous tissue. The cartilage showed metachromatic, perilacunar staini ng with toluidine blue and safronin O, alcian blue staining for chondr oitin and keratan sulfate, and intense immunostaining for type II coll agen. Osteocalcin was immunolocalized to the mineralized regions, cons istent with the formation of bone. Alkaline phosphatase was primarily observed in cell layers at boundaries between tissue types. Unstimulat ed monolayer cultures of PBMSC produced type I but not type II collage n, responded to dexamethasone (10(-8) M) with a 1.7-fold increase in a lkaline phosphatase activity, and were stimulated to divide by basic f ibroblast growth factor (1.5-fold; EC50 1 ng/ml). Transforming growth factor beta (TGF-beta) blocked both dexamethasone-induced alkaline pho sphatase expression (EC50 1 ng/ml of TGF-beta) and the mitogenic effec ts of bFGF (EC50 0.06 ng/ml of TGF-beta). When incubated for 10-14 day s in medium containing dexamethasone, beta-glycerophosphate and ascorb ate PBMSC formed mineralized nodules. Calcification occurred in the mi ddle of the aggregates and was associated with intensely alkaline phos phatase positive cells and a dense type I collagen-rich matrix. PBMSC also displayed colony-forming unit-fibroblastic activity, with approxi mately 1 in 80 of the plated cells formed colonies > 128 cells over 14 -21 days. PBMSC therefore mimic the known activities of stromal cells from other species, including the human, suggesting that they are a va lid model for skeletal research.