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Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium.

Kobayashi S, Takebe T, Zheng YW, Mizuno M, Yabuki Y, Maegawa J, Taniguchi H - PLoS ONE (2011)

Bottom Line: Furthermore, LRCs were successfully isolated and cultivated from auricular cartilage.Immunocytochemical analyses showed that LRCs express CD44 and integrin-α(5).Further characterization and utilization of the cell population should improve our understanding of basic cartilage biology and lead to advances in cartilage tissue engineering and novel therapeutic strategies for patients with craniofacial defects, including long-term tissue restoration.

View Article: PubMed Central - PubMed

Affiliation: Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Kanazawa-ku, Yokohama, Kanagawa, Japan.

ABSTRACT

Background: Based on evidence from several other tissues, cartilage stem/progenitor cells in the auricular cartilage presumably contribute to tissue development or homeostasis of the auricle. However, no definitive studies have identified or characterized a stem/progenitor population in mice auricle.

Methodology/principal findings: The 5-bromo-2'-deoxyuridine (BrdU) label-retaining technique was used to label dividing cells in fetal mice. Observations one year following the labeling revealed that label-retaining cells (LRCs) were present specifically in auricular perichondrium at a rate of 0.08±0.06%, but LRCs were not present in chondrium. Furthermore, LRCs were successfully isolated and cultivated from auricular cartilage. Immunocytochemical analyses showed that LRCs express CD44 and integrin-α(5). These LRCs, putative stem/progenitor cells, possess clonogenicity and chondrogenic capability in vitro.

Conclusions/significance: We have identified a population of putative cartilage stem/progenitor cells in the auricular perichondrium of mice. Further characterization and utilization of the cell population should improve our understanding of basic cartilage biology and lead to advances in cartilage tissue engineering and novel therapeutic strategies for patients with craniofacial defects, including long-term tissue restoration.

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In vitro characterization of CD44+ integrin-α5+ LRCs.(A) Immunocytochemistry of LRCs in vitro. Integrin-α5 and CD44 were co-expressed in BrdU-labeled cells. CD44 staining: scale bar = 20 µm; integrin-α5 staining: scale bar = 40 µm. Original magnification: ×200. (B) Colony formation of LRCs. Cells from 24-week-old mice, which were injected with BrdU as E17 to E19 fetuses, were harvested from mice auricle following collagenase digestion. Colony assay was performed to examine the clonogenicity of LRCs. Cells were stained at 1, 3 and 14 day after plated. Clonal colonies were stained with antibodies against DAPI, Ki-67 and BrdU. Scale bars = 20 µm or 100 µm (C) Alcian blue staining of cells isolated from auricular cartilage or dermal fibroblasts. Scale bar = 200 µm.
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pone-0026393-g005: In vitro characterization of CD44+ integrin-α5+ LRCs.(A) Immunocytochemistry of LRCs in vitro. Integrin-α5 and CD44 were co-expressed in BrdU-labeled cells. CD44 staining: scale bar = 20 µm; integrin-α5 staining: scale bar = 40 µm. Original magnification: ×200. (B) Colony formation of LRCs. Cells from 24-week-old mice, which were injected with BrdU as E17 to E19 fetuses, were harvested from mice auricle following collagenase digestion. Colony assay was performed to examine the clonogenicity of LRCs. Cells were stained at 1, 3 and 14 day after plated. Clonal colonies were stained with antibodies against DAPI, Ki-67 and BrdU. Scale bars = 20 µm or 100 µm (C) Alcian blue staining of cells isolated from auricular cartilage or dermal fibroblasts. Scale bar = 200 µm.

Mentions: Based on the immunohistochemistry analyses, we next examined whether auricular LRCs expressed CD44 and integrin-α5. Long-term BrdU labeled mice auricles were harvested at 24 weeks post birth. After enzymatic digestion of harvested auricle, we successfully isolated and cultivated the LRCs in vitro. Primary cultures of auricle cells isolated from 24-week-old BrdU-labeled mice were stained with antibodies against CD44 and integrin-α5. The long-term LRCs expressed both CD44 and integrin-α5, indicating both are potential stem cell markers (Figure 5A).


Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium.

Kobayashi S, Takebe T, Zheng YW, Mizuno M, Yabuki Y, Maegawa J, Taniguchi H - PLoS ONE (2011)

In vitro characterization of CD44+ integrin-α5+ LRCs.(A) Immunocytochemistry of LRCs in vitro. Integrin-α5 and CD44 were co-expressed in BrdU-labeled cells. CD44 staining: scale bar = 20 µm; integrin-α5 staining: scale bar = 40 µm. Original magnification: ×200. (B) Colony formation of LRCs. Cells from 24-week-old mice, which were injected with BrdU as E17 to E19 fetuses, were harvested from mice auricle following collagenase digestion. Colony assay was performed to examine the clonogenicity of LRCs. Cells were stained at 1, 3 and 14 day after plated. Clonal colonies were stained with antibodies against DAPI, Ki-67 and BrdU. Scale bars = 20 µm or 100 µm (C) Alcian blue staining of cells isolated from auricular cartilage or dermal fibroblasts. Scale bar = 200 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198405&req=5

pone-0026393-g005: In vitro characterization of CD44+ integrin-α5+ LRCs.(A) Immunocytochemistry of LRCs in vitro. Integrin-α5 and CD44 were co-expressed in BrdU-labeled cells. CD44 staining: scale bar = 20 µm; integrin-α5 staining: scale bar = 40 µm. Original magnification: ×200. (B) Colony formation of LRCs. Cells from 24-week-old mice, which were injected with BrdU as E17 to E19 fetuses, were harvested from mice auricle following collagenase digestion. Colony assay was performed to examine the clonogenicity of LRCs. Cells were stained at 1, 3 and 14 day after plated. Clonal colonies were stained with antibodies against DAPI, Ki-67 and BrdU. Scale bars = 20 µm or 100 µm (C) Alcian blue staining of cells isolated from auricular cartilage or dermal fibroblasts. Scale bar = 200 µm.
Mentions: Based on the immunohistochemistry analyses, we next examined whether auricular LRCs expressed CD44 and integrin-α5. Long-term BrdU labeled mice auricles were harvested at 24 weeks post birth. After enzymatic digestion of harvested auricle, we successfully isolated and cultivated the LRCs in vitro. Primary cultures of auricle cells isolated from 24-week-old BrdU-labeled mice were stained with antibodies against CD44 and integrin-α5. The long-term LRCs expressed both CD44 and integrin-α5, indicating both are potential stem cell markers (Figure 5A).

Bottom Line: Furthermore, LRCs were successfully isolated and cultivated from auricular cartilage.Immunocytochemical analyses showed that LRCs express CD44 and integrin-α(5).Further characterization and utilization of the cell population should improve our understanding of basic cartilage biology and lead to advances in cartilage tissue engineering and novel therapeutic strategies for patients with craniofacial defects, including long-term tissue restoration.

View Article: PubMed Central - PubMed

Affiliation: Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Kanazawa-ku, Yokohama, Kanagawa, Japan.

ABSTRACT

Background: Based on evidence from several other tissues, cartilage stem/progenitor cells in the auricular cartilage presumably contribute to tissue development or homeostasis of the auricle. However, no definitive studies have identified or characterized a stem/progenitor population in mice auricle.

Methodology/principal findings: The 5-bromo-2'-deoxyuridine (BrdU) label-retaining technique was used to label dividing cells in fetal mice. Observations one year following the labeling revealed that label-retaining cells (LRCs) were present specifically in auricular perichondrium at a rate of 0.08±0.06%, but LRCs were not present in chondrium. Furthermore, LRCs were successfully isolated and cultivated from auricular cartilage. Immunocytochemical analyses showed that LRCs express CD44 and integrin-α(5). These LRCs, putative stem/progenitor cells, possess clonogenicity and chondrogenic capability in vitro.

Conclusions/significance: We have identified a population of putative cartilage stem/progenitor cells in the auricular perichondrium of mice. Further characterization and utilization of the cell population should improve our understanding of basic cartilage biology and lead to advances in cartilage tissue engineering and novel therapeutic strategies for patients with craniofacial defects, including long-term tissue restoration.

Show MeSH
Related in: MedlinePlus