Sorting of Growth Plate Chondrocytes Allows the Isolation and Characterization of Cells of a Defined Differentiation Status

Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone-specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate. The RNA from femoral growth plates of 14 days old swiss white mice was cryosectioned and microdisected. The resulting destinct growth plate zones were used for RNA isolation via the PicoPure RNA isolation kit (Arcturus Bioscience, Mountain View, CA) and amplified via the MessageAMP aRNA kit (Ambion, Austin, TX). For microarray analysis, 1.25 μg of aRNA originating from each growth plate zone was mixed with 250 ng of random hexamer primers (Roche Diagnostics, Basel, Switzerland), heated at 70 °C for 10 min, and snap-cooled on ice. The oligo/aRNA mix was then employed for complementary DNA (cDNA) synthesis at 42 °C for 2 h in a total reaction volume of 30 μl using 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.2 mM dTTP, 0.3 mM amino-allyl dUTP (aa-dUTP) (Amersham Biosciences, Uppsala, Sweden), 10 mM DTT, 1x first strand buffer, and 150 units of SuperScript II (Invitrogen, Carlsbad, CA). The RNA templates were hydrolyzed at 65 °C for 15 min after addition 10 μl of 1 M NaOH. The mixture was then neutralized with 25 μl 1 M HEPES buffer pH 7.0 and unincorporated aa-dUTP and free amines were removed by applying and spinning the mixture onto QIAquick purification columns (Qiagen, Hilden, Germany). The columns were washed and Cy3 or Cy5 monoreactive dyes (Amersham Biosciences, Uppsala, Sweden) were resuspended in 15 μl 0.1 NaHCO3 pH 9.0 and pipetted directly onto the columns. The coupling reaction was left in the dark for one h at room temperature. The reactions were eluted with 80 μl of H2O and then diluted with 400 μl of PB buffer. To remove uncoupled dyes the Cy3- and Cy5-labeled cDNA of one two-color hybridization experiment were loaded sequentially onto one fresh QIAquick column, washed, and eluted with 30 μl of H2O. The labeled cDNA targets were volume-reduced to 16.2 μl in a vacuum centrifuge and stored in the dark at 4 °C for up to 1 h. Microarray slides printed with the 44K whole-genome microarrays (G4122A, Agilent Technologies, Santa Clara, CA) were pre-hybridized with 10 mg/ml BSA, 25% formamide, 5× SSC, 0.1% SDS for 45 min at 42 °C, rinsed twice in distilled H2O, and then air-dried. The cDNA targets were diluted to a final volume of 60 μl with 30 μl 2x hybridization buffer (50% formamide, 5× SSC, 0.2% SDS), 5 μl mouse Cot1 DNA (5 μg/μl), 3.8 μl polyA (10 mg/ml), and 5 μl of salmon sperm DNA (10 mg/ml). The samples were heated to 100 °C for 2 min and pipetted onto the microarray slides. The slides were covered with a 60 × 25 mm plastic cover slip (Schleicher und Schuell, Kassel, Gemany) and hybridization was conducted in sealed humid chambers at 42 °C for 17 h. The slides were washed serially in 1× SSC/0.2% SDS and 0.1x SSC/0.2% SDS for 5 min each and twice in 0.1× SCC for 2 min, spun dry for 5 min at 1000 ×g, and stored under dark conditions. The arrays were then scanned at 10 μm resolution on an Agilent scanner and the features acquired with the Feature extraction software.