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The transcription factor Sox2 is required for osteoblast self-renewal.

Basu-Roy U, Ambrosetti D, Favaro R, Nicolis SK, Mansukhani A, Basilico C - Cell Death Differ. (2010)

Bottom Line: In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability.Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression.These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, NYU School of Medicine, New York, NY, USA.

ABSTRACT
The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2- cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2- cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

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Sox2 is enriched in osteospheres and is required for osteosphere formation(A-D) Osteospheres from osteoblasts derived from P1 calvaria of wild type mice were collected by straining through a 40 μM strainer and RNA was extracted from the spheres. Gene expression of Sox2 (A), Osterix (B), Runx2 (C), and Collagen1-a1 (D) was analyzed by qRT-PCR using specific primers. All values are normalized to actin as an internal control and are expressed relative to total (adherent) cells in each case. * = p<0.05(E) Primary calvarial osteoblasts were plated in suspension culture in the presence or absence of 10 ng/ml recombinant FGF1. Spheres were counted after 7 days. * = p<0.05(F) Primary P1 calvarial osteoblasts from control (Sox2 flox/+;Cre, Sox2flox/- and Sox2flox/+) and cko (Sox2flox/-;Cre) littermates were plated in triplicate in 6 cm Corning ultra-low attachment plates. Total number of osteospheres was counted after 7 days. * = p < 0.05
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Figure 6: Sox2 is enriched in osteospheres and is required for osteosphere formation(A-D) Osteospheres from osteoblasts derived from P1 calvaria of wild type mice were collected by straining through a 40 μM strainer and RNA was extracted from the spheres. Gene expression of Sox2 (A), Osterix (B), Runx2 (C), and Collagen1-a1 (D) was analyzed by qRT-PCR using specific primers. All values are normalized to actin as an internal control and are expressed relative to total (adherent) cells in each case. * = p<0.05(E) Primary calvarial osteoblasts were plated in suspension culture in the presence or absence of 10 ng/ml recombinant FGF1. Spheres were counted after 7 days. * = p<0.05(F) Primary P1 calvarial osteoblasts from control (Sox2 flox/+;Cre, Sox2flox/- and Sox2flox/+) and cko (Sox2flox/-;Cre) littermates were plated in triplicate in 6 cm Corning ultra-low attachment plates. Total number of osteospheres was counted after 7 days. * = p < 0.05

Mentions: The results presented so far demonstrate that Sox2 is essential for osteoblast self-renewal and suggest that its expression may mark a population of uncommitted or unipotent osteoblasts. To investigate this hypothesis we have used a technique that has been successful in identifying such stem-like cells in the nervous system, mammary gland, skin and tumor tissues, i.e. the ability of these cells to form “spheres” of unattached cells in suspension culture. Indeed, a recent report has shown that calvarial osteoblasts are capable of forming “osteospheres” in suspension, representing progenitor/multipotent cell populations (28). In line with this observation, we found that osteoblasts isolated from calvaria of P1 pups are capable of forming osteospheres. We found that Sox2 expression is greatly enriched in the osteosphere fractions as compared to the total adherent cell population (Fig. 6A). On the other hand, the expression of OSX, Runx2, and ColI, markers of committed osteoblasts, was down-regulated in the spheres, confirming that they indeed represent a precursor cell population (Fig. 6B-D). Treatment with FGF, that induces Sox2 expression, increased osteosphere formation by primary osteoblasts 5-6 fold (Fig. 6E). As expected, we also found that osteoblasts made from calvaria of Sox2flox/-;Cre mice, that are a mosaic of Sox2 positive and Sox2 cells, show reduced osteosphere forming ability as compared to the osteoblasts isolated from control littermate mice, indicating that Sox2 is necessary to maintain a pool of osteoprogenitor cells (Figure 6F).


The transcription factor Sox2 is required for osteoblast self-renewal.

Basu-Roy U, Ambrosetti D, Favaro R, Nicolis SK, Mansukhani A, Basilico C - Cell Death Differ. (2010)

Sox2 is enriched in osteospheres and is required for osteosphere formation(A-D) Osteospheres from osteoblasts derived from P1 calvaria of wild type mice were collected by straining through a 40 μM strainer and RNA was extracted from the spheres. Gene expression of Sox2 (A), Osterix (B), Runx2 (C), and Collagen1-a1 (D) was analyzed by qRT-PCR using specific primers. All values are normalized to actin as an internal control and are expressed relative to total (adherent) cells in each case. * = p<0.05(E) Primary calvarial osteoblasts were plated in suspension culture in the presence or absence of 10 ng/ml recombinant FGF1. Spheres were counted after 7 days. * = p<0.05(F) Primary P1 calvarial osteoblasts from control (Sox2 flox/+;Cre, Sox2flox/- and Sox2flox/+) and cko (Sox2flox/-;Cre) littermates were plated in triplicate in 6 cm Corning ultra-low attachment plates. Total number of osteospheres was counted after 7 days. * = p < 0.05
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Related In: Results  -  Collection

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Figure 6: Sox2 is enriched in osteospheres and is required for osteosphere formation(A-D) Osteospheres from osteoblasts derived from P1 calvaria of wild type mice were collected by straining through a 40 μM strainer and RNA was extracted from the spheres. Gene expression of Sox2 (A), Osterix (B), Runx2 (C), and Collagen1-a1 (D) was analyzed by qRT-PCR using specific primers. All values are normalized to actin as an internal control and are expressed relative to total (adherent) cells in each case. * = p<0.05(E) Primary calvarial osteoblasts were plated in suspension culture in the presence or absence of 10 ng/ml recombinant FGF1. Spheres were counted after 7 days. * = p<0.05(F) Primary P1 calvarial osteoblasts from control (Sox2 flox/+;Cre, Sox2flox/- and Sox2flox/+) and cko (Sox2flox/-;Cre) littermates were plated in triplicate in 6 cm Corning ultra-low attachment plates. Total number of osteospheres was counted after 7 days. * = p < 0.05
Mentions: The results presented so far demonstrate that Sox2 is essential for osteoblast self-renewal and suggest that its expression may mark a population of uncommitted or unipotent osteoblasts. To investigate this hypothesis we have used a technique that has been successful in identifying such stem-like cells in the nervous system, mammary gland, skin and tumor tissues, i.e. the ability of these cells to form “spheres” of unattached cells in suspension culture. Indeed, a recent report has shown that calvarial osteoblasts are capable of forming “osteospheres” in suspension, representing progenitor/multipotent cell populations (28). In line with this observation, we found that osteoblasts isolated from calvaria of P1 pups are capable of forming osteospheres. We found that Sox2 expression is greatly enriched in the osteosphere fractions as compared to the total adherent cell population (Fig. 6A). On the other hand, the expression of OSX, Runx2, and ColI, markers of committed osteoblasts, was down-regulated in the spheres, confirming that they indeed represent a precursor cell population (Fig. 6B-D). Treatment with FGF, that induces Sox2 expression, increased osteosphere formation by primary osteoblasts 5-6 fold (Fig. 6E). As expected, we also found that osteoblasts made from calvaria of Sox2flox/-;Cre mice, that are a mosaic of Sox2 positive and Sox2 cells, show reduced osteosphere forming ability as compared to the osteoblasts isolated from control littermate mice, indicating that Sox2 is necessary to maintain a pool of osteoprogenitor cells (Figure 6F).

Bottom Line: In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability.Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression.These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, NYU School of Medicine, New York, NY, USA.

ABSTRACT
The development and maintenance of most tissues and organs require the presence of multipotent and unipotent stem cells that have the ability of self-renewal as well as of generating committed, further differentiated cell types. The transcription factor Sox2 is essential for embryonic development and maintains pluripotency and self-renewal in embryonic stem cells. It is expressed in immature osteoblasts/osteoprogenitors in vitro and in vivo and is induced by fibroblast growth factor signaling, which stimulates osteoblast proliferation and inhibits differentiation. Sox2 overexpression can by itself inhibit osteoblast differentiation. To elucidate its function in the osteoblastic lineage, we generated mice with an osteoblast-specific, Cre-mediated knockout of Sox2. These mice are small and osteopenic, and mosaic for Sox2 inactivation. However, culturing calvarial osteoblasts from the mutant mice for 2-3 passages failed to yield any Sox2- cells. Inactivation of the Sox2 gene by Cre-mediated excision in cultured osteoblasts showed that Sox2- cells could not survive repeated passage in culture, could not form colonies, and arrested their growth with a senescent phenotype. In addition, expression of Sox2-specific shRNAs in independent osteoblastic cell lines suppressed their proliferative ability. Osteoblasts capable of forming 'osteospheres' are greatly enriched in Sox2 expression. These data identify a novel function for Sox2 in the maintenance of self-renewal in the osteoblastic lineage.

Show MeSH
Related in: MedlinePlus