Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells.
BACKGROUND
The objective of this study was to isolate osteoprogenitor cells (OPC) from BM mesenchymal stromal cells (MSC) and test their capacity to proliferate and differentiate into osteoblasts.


METHODS
Human MSC were separated on a Percoll gradient and cultured in DMEM supplemented with 15% human serum, and characterized by flow cytometric analyzes for CD34, CD13, CD90, CD105 and CD117. To induce differentiation, cultured cells were exposed to 10(-7) m dexamethasone (dexa) and/or 10(-3) m sodium beta-glycerophosphate (beta-GlyP) and 1,25-dihydroxyvitamin D3 (calcitriol) or 9-cis-retinoic acid (9-RA).


RESULTS
alkaline phosphatase (AP) activity was detected in cells irrespective of the dexa and/or beta-GlyP treatment. Antigenic phenotypes of MSC were CD34- (more than 99%) and CD13+ CD90+ CD105+ CD117+ (c. 50%). The treatment induced extracellular calcium deposition and gene and protein expression of osteonectin (ON) and bone sialoprotein (BSP): beta-GlyP induced an increase (c. 2.2-fold) of the ON gene and dexa augmented (c. 2.7-fold) the gene expression of BSP II. Gene expression of BSP I reached a maximum at 3 weeks of combined treatment. Osteocalcin gene expression was induced only after additional treatment with calcitriol or 9-RA. Ultrastructural analysis revealed the secretory phenotype of OPC.


CONCLUSIONS
Under appropriate treatment, MSC can give rise to OPC that have the capacity to differentiate into osteoblasts characterized by the expression of osteogenic markers, osteoblastic properties and stromal BM cells phenotypes. These cells may represent a promising material to be utilized in orthopedic cellular therapy.
Titorencu
I
I
Department of Cell Culture, Institute of Cellular Biology and Pathology, Nicolae Simionescu, Bucharest, Romania.
Jinga
V  V
V
Constantinescu
E
E
Gafencu
A  V
A
Ciohodaru
C
C
Manolescu
I
I
Zaharia
C
C
Simionescu
M
M
D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism; D015703:Antigens, CD / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology* / Q000378:metabolism; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D049109:Cell Proliferation; D017077:Culture Media, Conditioned; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D010012:Osteogenesis; D015676:Osteonectin / Q000302:isolation & purification / Q000378:metabolism; D012795:Sialoglycoproteins / Q000302:isolation & purification / Q000378:metabolism
D015703:Antigens, CD; D017077:Culture Media, Conditioned; C546871:IBSP protein, human; D058574:Integrin-Binding Sialoprotein; D015676:Osteonectin; D012795:Sialoglycoproteins; D000469:Alkaline Phosphatase