Osteogenic differentiation of mouse embryonic stem cells: differential gene expression analysis by cDNA microarray and purification of osteoblasts by cadherin-11 magnetically activated cell sorting.
We have previously shown osteogenic differentiation of mouse embryonic stem (ES) cells and temporal enrichment with osteoblastic cells, by stimulation with serum-containing culture medium supplemented with beta-glycerophosphate, ascorbate, and dexamethasone. In our present study we have used similar culture conditions to further investigate osteogenic differentiation of mouse ES cells. Using reverse transcription-polymerase chain reaction (RT-PCR) we demonstrated the expression of genes associated with osteoblast differentiation including the bone matrix protein osteocalcin and the transcription factor Cbfa-1/runx2. Furthermore, results of cDNA microarray analysis, and subsequent RT-PCR analysis of differentiating ES cells after exposure to osteogenic stimuli, revealed a combination of upregulation of genes involved in osteoblast differentiation including osteopontin, HSP-47, and IGF-II coupled with downregulation of genes involved in differentiation of other phenotypes such as the neuroectoderm factor Stra-13. Finally, we have applied magnetically activated cell-sorting methods to ES cell cultures treated with osteogenic stimuli and, using an antibody to cadherin-11, have purified a subpopulation of cells with osteoblastic characteristics.
Bourne
S
S
Tissue Engineering and Regenerative Medicine Centre, Cell Biology Laboratories, Imperial College, Faculty of Medicine, Chelsea and Westminster Hospital Campus, London, UK.
Polak
J M
JM
Hughes
S P F
SP
Buttery
L D K
LD
D000818:Animals; D015820:Cadherins; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D018189:Immunomagnetic Separation / Q000379:methods*; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology*; D023822:Tissue Engineering / Q000379:methods*
D015820:Cadherins; C086960:osteoblast cadherin