Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

Abstract

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. ß

Novel type II collagen reporter mice: New tool for assessing collagen 2α1 expression in vivo and in vitro

“…The resulting primary murine chondrocytes are mostly proliferative chondrocytes, which may be more appropriate for in vitro studies of growth plate biology than chondrocytes harvested from rib epiphyseal cartilage, which contains mainly to reserve, i.e., quiescent, chondrocytes. Zone-specific markers have recently been employed to sort enriched chondrocyte populations in respect of their differentiation stage (Belluoccio et al, 2010). This approach and our use of pd2EGFP to harvest growth plate chondrocytes complement each other well.…”

Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes via repression of Sox9

“…Hypertrophic cartilage was microdissected from P5 long bones and ribs, and digested with collagenase as described (Belluoccio et al, 2010). Total RNA was extracted using TRIzol (Life Technologies) and digested with DNase I to eliminate any contaminating genomic DNA.…”

Phenotypic Diversity in Chondromyxoid Fibroma Reveals Differentiation Pattern of Tumor Mimicking Fetal Cartilage Canals Development

“…Furthermore, the secondary ossification centers within the femoral head occur substantially later in the postnatal development of the hip. [23][24] Moreover, the chondrocytes in chondromyxoid fibroma neither form columnar structures nor display voluminous clear cytoplasm characteristic for the hypertrophic chondrocytes [25][26][27] occurring typically within the growth plate cartilage. Indeed, we are not aware of any report on chondromyxoid fibroma with columnar organized or hypertrophic appearing chondrocyte proliferations.…”