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The suture provides a niche for mesenchymal stem cells of craniofacial bones.

Zhao H, Feng J, Ho TV, Grimes W, Urata M, Chai Y - Nat. Cell Biol. (2015)

Bottom Line: Gli1+ cells are typical MSCs in vitro.Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating that these cells are an indispensable stem cell population.Twist1(+/-) mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA 103 Los Angeles, California 90033, USA.

ABSTRACT
Bone tissue undergoes constant turnover supported by stem cells. Recent studies showed that perivascular mesenchymal stem cells (MSCs) contribute to the turnover of long bones. Craniofacial bones are flat bones derived from a different embryonic origin than the long bones. The identity and regulating niche for craniofacial-bone MSCs remain unknown. Here, we identify Gli1+ cells within the suture mesenchyme as the main MSC population for craniofacial bones. They are not associated with vasculature, give rise to all craniofacial bones in the adult and are activated during injury repair. Gli1+ cells are typical MSCs in vitro. Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating that these cells are an indispensable stem cell population. Twist1(+/-) mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells. Our study indicates that craniofacial sutures provide a unique niche for MSCs for craniofacial bone homeostasis and repair.

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Gli1+ cells are MSCs in vitro. (a-f) Immunohistochemical staining of MSC markers CD73 (a), CD90 (b), CD44 (c), CD146 (d) and Sca1 (e) in the suture mesenchyme of Gli1-LacZ mice. Sca1 expression is also detectable in the periosteum (f). Arrows indicate expression. Dotted lines outline bone margins. (g) FACS analysis of suture mesenchymal cells harvested from one-month-old Gli1-CE;R26Tdtomato mice induced with tamoxifen. (h-l) Gli1+ cells form clones in culture. Positive clones were picked based on their fluorescence (k). Alizarin red (i), Alcian blue (j), and perilipin (l) staining indicates that cells from single clones can undergo tri-lineage osteogenic (osteo), chondrogenic (chondro), and adipogenic (adipo) differentiation. (m) Quantitation of the fraction of differentiated cells in the suture MSC culture normalized to that of BMMSCs under the same conditions. Values are plotted as mean ±SEM. *, student t-test, p=0.02, n=5 cultures derived from different mice. Scale bars, 100 μm.
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Figure 4: Gli1+ cells are MSCs in vitro. (a-f) Immunohistochemical staining of MSC markers CD73 (a), CD90 (b), CD44 (c), CD146 (d) and Sca1 (e) in the suture mesenchyme of Gli1-LacZ mice. Sca1 expression is also detectable in the periosteum (f). Arrows indicate expression. Dotted lines outline bone margins. (g) FACS analysis of suture mesenchymal cells harvested from one-month-old Gli1-CE;R26Tdtomato mice induced with tamoxifen. (h-l) Gli1+ cells form clones in culture. Positive clones were picked based on their fluorescence (k). Alizarin red (i), Alcian blue (j), and perilipin (l) staining indicates that cells from single clones can undergo tri-lineage osteogenic (osteo), chondrogenic (chondro), and adipogenic (adipo) differentiation. (m) Quantitation of the fraction of differentiated cells in the suture MSC culture normalized to that of BMMSCs under the same conditions. Values are plotted as mean ±SEM. *, student t-test, p=0.02, n=5 cultures derived from different mice. Scale bars, 100 μm.

Mentions: The current definition of MSCs is based upon in vitro assays. Therefore, we evaluated Gli1+ cells in accordance with these standards. Immunohistochemical staining indicated that the majority of Gli1+ cells in the suture mesenchyme do not express MSC markers including CD90, CD73, CD44, Sca1, and CD146 (Figure 4a-f). CD44 expression was detectable in the osteogenic front region (Figure 4c), and Sca1 expression was detectable in the periosteum (Figure 4f). To study their in vitro properties, Gli1-CreERT2;R26Tdtomatoflox mice at 1 month of age were induced with tamoxifen. Mesenchymal cells were obtained from the suture one week after induction and cultured. FACS analysis indicated that Gli1+ cells and their derivatives express high levels of typical MSC markers including CD44, CD90, Sca1, CD146, and CD73, but not CD34 (Figure 4g). Clonal culture indicated that Gli1+ cells possess clone-forming ability (Figure 4h, k). Subculture of clones indicated that clones derived from single Gli1+ cells are capable of osteogenic, chondrogenic and adipogenic differentiation (Figure 4i-l) and are therefore multipotent31. We compared the differentiation ability of suture MSCs with MSCs taken from femur bone marrow of the same mice. Although the osteogenic and chondrogenic differentiation abilities were comparable, the adipogenic differentiation ability of the suture MSCs was much weaker than that of the bone marrow MSCs (Figure 4m). These data indicate that although Gli1+ cells do not express MSC markers in vivo, they behave like typical MSCs in vitro and are more committed towards osteochondrogenic lineages. Traditional MSC markers routinely used to identify MSCs in vitro are not ideal markers for MSCs in vivo.


The suture provides a niche for mesenchymal stem cells of craniofacial bones.

Zhao H, Feng J, Ho TV, Grimes W, Urata M, Chai Y - Nat. Cell Biol. (2015)

Gli1+ cells are MSCs in vitro. (a-f) Immunohistochemical staining of MSC markers CD73 (a), CD90 (b), CD44 (c), CD146 (d) and Sca1 (e) in the suture mesenchyme of Gli1-LacZ mice. Sca1 expression is also detectable in the periosteum (f). Arrows indicate expression. Dotted lines outline bone margins. (g) FACS analysis of suture mesenchymal cells harvested from one-month-old Gli1-CE;R26Tdtomato mice induced with tamoxifen. (h-l) Gli1+ cells form clones in culture. Positive clones were picked based on their fluorescence (k). Alizarin red (i), Alcian blue (j), and perilipin (l) staining indicates that cells from single clones can undergo tri-lineage osteogenic (osteo), chondrogenic (chondro), and adipogenic (adipo) differentiation. (m) Quantitation of the fraction of differentiated cells in the suture MSC culture normalized to that of BMMSCs under the same conditions. Values are plotted as mean ±SEM. *, student t-test, p=0.02, n=5 cultures derived from different mice. Scale bars, 100 μm.
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Figure 4: Gli1+ cells are MSCs in vitro. (a-f) Immunohistochemical staining of MSC markers CD73 (a), CD90 (b), CD44 (c), CD146 (d) and Sca1 (e) in the suture mesenchyme of Gli1-LacZ mice. Sca1 expression is also detectable in the periosteum (f). Arrows indicate expression. Dotted lines outline bone margins. (g) FACS analysis of suture mesenchymal cells harvested from one-month-old Gli1-CE;R26Tdtomato mice induced with tamoxifen. (h-l) Gli1+ cells form clones in culture. Positive clones were picked based on their fluorescence (k). Alizarin red (i), Alcian blue (j), and perilipin (l) staining indicates that cells from single clones can undergo tri-lineage osteogenic (osteo), chondrogenic (chondro), and adipogenic (adipo) differentiation. (m) Quantitation of the fraction of differentiated cells in the suture MSC culture normalized to that of BMMSCs under the same conditions. Values are plotted as mean ±SEM. *, student t-test, p=0.02, n=5 cultures derived from different mice. Scale bars, 100 μm.
Mentions: The current definition of MSCs is based upon in vitro assays. Therefore, we evaluated Gli1+ cells in accordance with these standards. Immunohistochemical staining indicated that the majority of Gli1+ cells in the suture mesenchyme do not express MSC markers including CD90, CD73, CD44, Sca1, and CD146 (Figure 4a-f). CD44 expression was detectable in the osteogenic front region (Figure 4c), and Sca1 expression was detectable in the periosteum (Figure 4f). To study their in vitro properties, Gli1-CreERT2;R26Tdtomatoflox mice at 1 month of age were induced with tamoxifen. Mesenchymal cells were obtained from the suture one week after induction and cultured. FACS analysis indicated that Gli1+ cells and their derivatives express high levels of typical MSC markers including CD44, CD90, Sca1, CD146, and CD73, but not CD34 (Figure 4g). Clonal culture indicated that Gli1+ cells possess clone-forming ability (Figure 4h, k). Subculture of clones indicated that clones derived from single Gli1+ cells are capable of osteogenic, chondrogenic and adipogenic differentiation (Figure 4i-l) and are therefore multipotent31. We compared the differentiation ability of suture MSCs with MSCs taken from femur bone marrow of the same mice. Although the osteogenic and chondrogenic differentiation abilities were comparable, the adipogenic differentiation ability of the suture MSCs was much weaker than that of the bone marrow MSCs (Figure 4m). These data indicate that although Gli1+ cells do not express MSC markers in vivo, they behave like typical MSCs in vitro and are more committed towards osteochondrogenic lineages. Traditional MSC markers routinely used to identify MSCs in vitro are not ideal markers for MSCs in vivo.

Bottom Line: Gli1+ cells are typical MSCs in vitro.Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating that these cells are an indispensable stem cell population.Twist1(+/-) mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Craniofacial Molecular Biology, Ostrow School of Dentistry, University of Southern California, 2250 Alcazar Street, CSA 103 Los Angeles, California 90033, USA.

ABSTRACT
Bone tissue undergoes constant turnover supported by stem cells. Recent studies showed that perivascular mesenchymal stem cells (MSCs) contribute to the turnover of long bones. Craniofacial bones are flat bones derived from a different embryonic origin than the long bones. The identity and regulating niche for craniofacial-bone MSCs remain unknown. Here, we identify Gli1+ cells within the suture mesenchyme as the main MSC population for craniofacial bones. They are not associated with vasculature, give rise to all craniofacial bones in the adult and are activated during injury repair. Gli1+ cells are typical MSCs in vitro. Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating that these cells are an indispensable stem cell population. Twist1(+/-) mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells. Our study indicates that craniofacial sutures provide a unique niche for MSCs for craniofacial bone homeostasis and repair.

Show MeSH
Related in: MedlinePlus