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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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48-week chase analysis of BrdU-labeled cells and Ki67-positive cells.BrdU-labeled cells gradually decreased in auricular cartilage (White arrowheads). A few BrdU-labeled cells were present in the perichondrium of 48-week-old, BrdU-labeled offspring. No LRCs were observed in chondrocytes of the chondrium 4 weeks after BrdU labeling. No Ki67-positive cells were seen in perichondrium 1 week following birth (White arrows). No Ki67-positive cells were observed in the chondrium 2 weeks following birth. (A) 0 day, (B) 3 day, (C) 1 week, (D) 2 weeks, (E) 4 weeks, (F) 24 weeks and (G) 48 weeks. From the left, Alcian blue staining, DAPI, BrdU, Ki67, and a merged image. Two-headed arrows: the cartilage width including perichondrium. Black scale bar = 200 µm, White scale bar = 50 µm.
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pone-0026393-g002: 48-week chase analysis of BrdU-labeled cells and Ki67-positive cells.BrdU-labeled cells gradually decreased in auricular cartilage (White arrowheads). A few BrdU-labeled cells were present in the perichondrium of 48-week-old, BrdU-labeled offspring. No LRCs were observed in chondrocytes of the chondrium 4 weeks after BrdU labeling. No Ki67-positive cells were seen in perichondrium 1 week following birth (White arrows). No Ki67-positive cells were observed in the chondrium 2 weeks following birth. (A) 0 day, (B) 3 day, (C) 1 week, (D) 2 weeks, (E) 4 weeks, (F) 24 weeks and (G) 48 weeks. From the left, Alcian blue staining, DAPI, BrdU, Ki67, and a merged image. Two-headed arrows: the cartilage width including perichondrium. Black scale bar = 200 µm, White scale bar = 50 µm.

Mentions: Then, we treated pregnant mice with BrdU on day17 to 19 of gestation to label proliferating cells in the fetuses during auricular development. The auricles of the offspring were analyzed at multiple time points (on day 0 and day 3 and at 1, 2, 4, 24, and 48 weeks) (Figure 2A–G). To assess the efficiency of BrdU incorporation, the auricles of neonate mice were immunohistochemically labeled with monoclonal antibodies against BrdU; the majority of cells (83.6±5.4%) were labeled in neonates (N = 6) (Figure 3A). Within two weeks, the number of BrdU-positive cells, i.e., label-retaining cells (LRCs), rapidly decreased to 2.5±0.2% (N = 6). Concomitantly, localization of LRCs is restricted to the perichondrium layer (50.0%, 53.8%, 80.3% and 69.7% of BrdU positive cells, respectively)(Figure 3B). After 4 weeks, no LRCs were observed in the chondrium. However, LRCs were observed in the perichondrium even after 4 weeks (0.3±0.2% of cells) (N = 3). After 24 and 48 weeks, long-term LRCs were detected only in the perichondrium (0.1±0.05% and 0.08±0.06% of all 4,6-diamidino-2-phenylindole (DAPI)-stained cells, respectively) (N = 3, each timepoint) (Figure S2A, B, and Figure 3B). These LRCs, observed only in the perichondrium layer, seemed dormant, which is characteristic of putative stem cells.


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)

48-week chase analysis of BrdU-labeled cells and Ki67-positive cells.BrdU-labeled cells gradually decreased in auricular cartilage (White arrowheads). A few BrdU-labeled cells were present in the perichondrium of 48-week-old, BrdU-labeled offspring. No LRCs were observed in chondrocytes of the chondrium 4 weeks after BrdU labeling. No Ki67-positive cells were seen in perichondrium 1 week following birth (White arrows). No Ki67-positive cells were observed in the chondrium 2 weeks following birth. (A) 0 day, (B) 3 day, (C) 1 week, (D) 2 weeks, (E) 4 weeks, (F) 24 weeks and (G) 48 weeks. From the left, Alcian blue staining, DAPI, BrdU, Ki67, and a merged image. Two-headed arrows: the cartilage width including perichondrium. Black scale bar = 200 µm, White scale bar = 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198405&req=5

pone-0026393-g002: 48-week chase analysis of BrdU-labeled cells and Ki67-positive cells.BrdU-labeled cells gradually decreased in auricular cartilage (White arrowheads). A few BrdU-labeled cells were present in the perichondrium of 48-week-old, BrdU-labeled offspring. No LRCs were observed in chondrocytes of the chondrium 4 weeks after BrdU labeling. No Ki67-positive cells were seen in perichondrium 1 week following birth (White arrows). No Ki67-positive cells were observed in the chondrium 2 weeks following birth. (A) 0 day, (B) 3 day, (C) 1 week, (D) 2 weeks, (E) 4 weeks, (F) 24 weeks and (G) 48 weeks. From the left, Alcian blue staining, DAPI, BrdU, Ki67, and a merged image. Two-headed arrows: the cartilage width including perichondrium. Black scale bar = 200 µm, White scale bar = 50 µm.
Mentions: Then, we treated pregnant mice with BrdU on day17 to 19 of gestation to label proliferating cells in the fetuses during auricular development. The auricles of the offspring were analyzed at multiple time points (on day 0 and day 3 and at 1, 2, 4, 24, and 48 weeks) (Figure 2A–G). To assess the efficiency of BrdU incorporation, the auricles of neonate mice were immunohistochemically labeled with monoclonal antibodies against BrdU; the majority of cells (83.6±5.4%) were labeled in neonates (N = 6) (Figure 3A). Within two weeks, the number of BrdU-positive cells, i.e., label-retaining cells (LRCs), rapidly decreased to 2.5±0.2% (N = 6). Concomitantly, localization of LRCs is restricted to the perichondrium layer (50.0%, 53.8%, 80.3% and 69.7% of BrdU positive cells, respectively)(Figure 3B). After 4 weeks, no LRCs were observed in the chondrium. However, LRCs were observed in the perichondrium even after 4 weeks (0.3±0.2% of cells) (N = 3). After 24 and 48 weeks, long-term LRCs were detected only in the perichondrium (0.1±0.05% and 0.08±0.06% of all 4,6-diamidino-2-phenylindole (DAPI)-stained cells, respectively) (N = 3, each timepoint) (Figure S2A, B, and Figure 3B). These LRCs, observed only in the perichondrium layer, seemed dormant, which is characteristic of putative stem cells.

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