Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation.
Mesenchymal stem cells give rise to osteoprogenitors that proliferate and differentiate into identifiable preosteoblasts, osteoblasts, bone lining cells and osteocytes. To identify and establish a molecular profile for the more primitive and uncharacterized cells in the lineage, relatively rare (<1%) osteoprogenitors present in primary cultures of fetal rat calvaria cell populations were identified by a replica plating technique. Since the cell number was limited in each colony sampled, we used global amplification PCR to analyze the repertoire of genes expressed in osteoprogenitors. We established a molecular fingerprint and a developmental sequence based on simultaneous expression patterns for both known osteoblast-associated markers (collagen type I, alkaline phosphatase, osteopontin, bone sialoprotein, PTH1R and osteocalcin) and potential regulatory molecules (i.e. FGFR1, PDGF-Ralpha and PTHrP). By analysis of 99 osteoprogenitor and osteoblast colonies captured by replica plating at different developmental stages, we found: (1) a recognizable cohort of cells considered more primitive than committed osteoprogenitors; (2) a cohort of early progenitors transiently expressing bone sialoprotein; and (3) that mRNAs for FGF-R1, PDGF-Ralpha and PTH1R were expressed earlier than other markers and tended to increase and decrease in relative concert with the osteoblast-specific markers. The observations suggest that within the osteoblast differentiation sequence both discrete stages and continua of changing marker expression levels occur with variation in expression for any given marker. This combined approach of replica plating and global amplification PCR allows molecular fingerprinting of definitive primitive osteoprogenitors and will aid in identifying novel developmental stages and novel differentiation stage-specific genes as these cells progress through their differentiation sequence.
Liu
Fina
F
Department of Anatomy & Cell Biology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
Malaval
Luc
L
Aubin
Jane E
JE
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D058574:Integrin-Binding Sialoprotein; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics; D053495:Osteopontin; D044162:Parathyroid Hormone-Related Protein / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D051496:Receptor, Fibroblast Growth Factor, Type 1; D020796:Receptor, Platelet-Derived Growth Factor alpha / Q000235:genetics; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics; D012795:Sialoglycoproteins / Q000235:genetics; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism*
D024042:Collagen Type I; D018076:DNA, Complementary; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D044162:Parathyroid Hormone-Related Protein; D012333:RNA, Messenger; D017468:Receptors, Fibroblast Growth Factor; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D015675:Osteocalcin; D053495:Osteopontin; C496350:Fgfr1 protein, rat; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D020796:Receptor, Platelet-Derived Growth Factor alpha; D000469:Alkaline Phosphatase