Reciprocal control of osteoblast/chondroblast and osteoblast/adipocyte differentiation of multipotential clonal human marrow stromal F/STRO-1+ cells

Abstract

The regulation of human bone marrow stromal precursor cell differentiation toward the chondrocyte, osteoblast or adipocyte lineages is not known. In this study, we assessed the lineage-specific differentiation and conversion of immortalized clonal F/STRO-1(+) A human fetal bone marrow stromal cells under the control of dexamethasone (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F/STRO-1(+) A cells expressed markers mRNAs or proteins of the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline phosphatase (ALP), type 1 collagen], of the chondrocyte lineage (aggrecan, types 2, 9 and 10 collagen), and of the adipocyte lineage (PPARgamma2, C/EBPalpha, aP2, G3PDH, lipoprotein lipase, leptin). Treatment with Dex increased CBFA1, OC and ALP mRNA and protein levels. Exposure to LA enhanced expression of adipocytic genes and cytoplasmic triglycerides accumulation, and suppressed the Dex-induced stimulation of osteoblast marker genes. Indo/Ins stimulated the synthesis of aggrecan and type 2 collagen and increased types 9 and 10 collagen mRNA levels, and suppressed both basal and Dex-promoted expression of osteoblast markers. Conversely, stimulation of osteoblastogenesis by Dex suppressed both basal and Indo/Ins-stimulated chondrocyte genes. Thus, the clonal human fetal bone marrow stromal F/STRO-1(+) A cell line is a lineage-unrestricted common progenitor that expresses tripotential adipocyte, osteoblast or chondrocyte characteristics. Our data also show that differentiation towards one pathway in response to Dex, Indo/Ins and LA restricts expression of other lineage-specific genes, and provide evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of clonal F/STRO-1(+) human bone marrow stromal cells.

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

“…At the indicated time points, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline. Matrix mineralization was evaluated by alizarin red and von Kossa staining as described (44) and microphotographed using an Olympus microscope (Japan). Glycoprotein synthesis was determined by alcian blue staining (44).…”

“…Matrix mineralization was evaluated by alizarin red and von Kossa staining as described (44) and microphotographed using an Olympus microscope (Japan). Glycoprotein synthesis was determined by alcian blue staining (44). For adipogenesis, the accumulation of lipid droplets, a hallmark of functional adipogenesis, was detected by oil red staining (44) and quantified at 14 days of culture, as described (45).…”

“…Glycoprotein synthesis was determined by alcian blue staining (44). For adipogenesis, the accumulation of lipid droplets, a hallmark of functional adipogenesis, was detected by oil red staining (44) and quantified at 14 days of culture, as described (45). Briefly, the stain solution was removed, and cells were rinsed in 500 l of 60% isopropyl alcohol for 5 s. To extract dye, 700 l of 60% isopropyl alcohol was added per well, and sealed plates were shaken for 2 h. The extracted dye was quantitated spectrophotometrically at 510 nm.…”

Promotion of Osteoblast Differentiation in Mesenchymal Cells Through Cbl-Mediated Control of STAT5 Activity

“…Additionally, we used human clonal pluripotent bone marrow stromal cells immunoselected with the murine IgM monoclonal antibody STRO-1, a cell surface antigen expressed by stromal elements in human bone marrow [39]. The selected cells (F/STRO-1 þ A) show osteogenic differentiation potential under appropriate stimulation [2,40]. Cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Paisley, Scotland, http://www.invitrogen.com) supplemented with 10% heat inactivated fetal calf serum, 1% L-glutamine, and penicillin/streptomycin (10,000 U/mL and 10,000 lg/mL, respectively) at 37 C in humidified atmosphere containing 5% CO 2 in air.…”

Cell Therapy for Bone Disease: A Review of Current Status

“…According to that model of differentiation, the osteogenic pathway is the default lineage of this population. Experiments have been performed where the reciprocal control of osteogenic-chondrogenic and osteogenicadipogenic differentiation has been demonstrated in an immortalized cell line of human stromal cells [18].…”