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

Abstract

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.

Comparison of Osteogenic Differentiation of Embryonic Stem Cells and Primary Osteoblasts Revealed by Responses to IL-1β, TNF-α, and IFN-γ

“…Osteogenesis of ESCs often requires first inducing ES-derived MSCs [71,72], but the intention of this study was to avoid the need to perform this step and directly harvest a population of osteogenic cells from the mESC cultures. Cad-11 has previously been used to sort osteogenic cells within differentiating ESC-derived cultures [18]. In our cell sorting experiments, we achieved a cad-11-positive population of about 21%, which was considerably less than previously described [18].…”

“…In vitro osteogenic differentiation of primary osteoblasts [8][9][10][11][12][13]44] and ESCs [17][18][19][20][21] have both been well described, but there have been few comparative studies. In this study, both cell types showed expression of markers indicative of osteogenic differentiation and formed nodules comprising Values corrected to proportion of osteogenic control.…”

“…Both mouse [17,18] and human ESCs [19][20][21] have been shown to display the features of osteogenically differentiated cells in vitro, exhibiting molecular and structural features resembling bone tissue by the formation of mineralized bone nodule structures. The majority of osteogenic protocols for ESCs direct cell differentiation by including factors in the culture medium, such as b-glycerophosphate (BGP), ascorbate, dexamethasone, simvastatin, retinoic acid, vitamin D 3 , and bone morphogenic proteins [3,[22][23][24][25][26][27][28][29][30].…”

“…In this study, we investigate cadherin-11 (cad-11) as a marker for the purification of osteogenically differentiated ESCs. Cad-11 has previously been used to purify ESCs [18] and is a cell adhesion molecule strongly associated with bone formation and osteogenic differentiation [39,40].…”

Expression of the extracellular domain of OB-cadherin as an Fc fusion protein using bicistronic retroviral expression vector

“…Expression of OB-cadherin is associated with osteoblast differentiation and has been proposed to function in cell sorting, migration, and alignment during the maturation of osteoblasts [12]. As a result, OB-cadherin has also been used as a marker for the selection of osteoblastic lineage cells from embryonic stem cells induced to differentiate into various lineages [13].…”