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

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.

Figure 1. Development of murine external ears.

(A) The external ear at several developmental stages. From the left, ears were photographed 3 days, 1 week, 2 weeks, 4 weeks, and 48 weeks after birth. (B) Mean surface area of the external ear and mean thickness of auricular cartilages changed during postnatal development. * ; 3 days, ; mean surface area of the external ear (mm2), ; mean thickness of auricular cartilage in the middle area of the external ear (µm) (N = 6).

Figure 2. 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.

Figure 3. Presence of long-term label retaining cells in auricuar perichondrium 48 weeks following birth.

(A) Quantification of BrdU-label retaining cells from 0 day to 48 weeks in mice auricular cartilage (N = 6). (B) The distributions of LRCs between chondrium and perichondrium layer. 4 weeks after birth, all LRCs were detected in perichondrium, but not chondrium layer. (C) Proliferation of cells in auricular cartilage. Cells in both layers extensively proliferate till 2 weeks post birth. Graph shows time course-dependent changes of Ki67-positive cell index (KI) in perichondrium and chondrium for 48 weeks after birth. ;KI of Perichondrium, ; KI of Chondrium.

Figure 4. Localization of long-term LRCs in different parts of the external ear.

(A) A few LRCs were present in the perichondrium in the distal and middle areas of the external ear (White arrowheads). Many LRCs were present in the opening of the external acoustic meatus (White arrowheads). Two-headed arrows: Chondrium. Scale bar = 50 µm. Original magnification: ×40 (Alcian blue staining), ×100 (BrdU staining). (B) The LRCs of the distal and middle areas of the external ear and in the opening of the external acoustic meatus. The LRCs in the opening of the external acoustic meatus was much higher than that of the distal and middle areas (N = 6).

Figure 5. 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.