EdU-Based Assay of Cell Proliferation and Stem Cell Quiescence in Skeletal Tissue Sections

Abstract

Identifying and tracking proliferating and quiescent cells in situ is an important phenotyping component of skeletal tissues in development, physiology and disease. Among all the methods that exist, which include immunostaining for cell cycle-specific proteins, the gold standards use thymidine analogs. These compounds label proliferating cells by being incorporated into de novo-synthesized genomic DNA. 5-bromo-2'-deoxyuridine (BrdU) has traditionally been used for this purpose, but its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to DNA. An alternative, more recently developed, uses 5-ethynyl-2'-deoxyuridine (EdU). This thymidine analog is detected by click chemistry, that is, covalent cross-linking of its ethynyl group with a fluorescent azide that is small enough to easily penetrate native tissues and reach DNA. In addition to being simple and quick, this EdU-based assay is compatible with other protocols, such as immunostaining, on the same tissue sections. We here describe an EdU-based protocol optimized to label and functionally assess actively proliferating cells as well as slowly dividing cells, including stem cells, in mouse skeletal tissues.

Keywords: Bone; BrdU; Cartilage; Cell labeling; Cell proliferation; EdU; In situ; Skeleton; Stem cell.

Fig. 1

Schematic of the major steps involved in the EdU-based assay of actively proliferating and slowly dividing/stem cells. See Subheadings 1 and 2

Fig. 2

EdU assay in articular and growth plate cartilage of juvenile mice illustrating the differences in results obtained when one or several of EdU injections are performed and when short or extended periods of time are applied between cell labeling and tissue analysis. Mice received a single injection of EdU (on postnatal day 14) and were euthanized 2 h, 2 days, 7 days, or 12 days later, or received EdU on three consecutive days (on postnatal days 22, 23, and 24) and were euthanized 12 days after the last injection. Sagittal knee sections were stained for EdU (green signal) and with DAPI (blue signal). The top-row pictures show that articular chondrocytes have a very low rate of proliferation. Only a few cells incorporated EdU in 2 h (left). Cell doublets are seen 2, 7, and 12 days after EdU injection and their numbers are similar to those of single cells after 2 h, indicating that the cells underwent one division within 2 days after labeling and none in the next 10 days. Note that most EdU-positive cells are located in the superficial layers of articular cartilage and gave rise to progeny aligned with the articular surface (increasing the tissue surface), and that most other EdU-positive cells are located in the upper half of the tissue (non-mineralized cartilage) and proliferated in columns perpendicular to the tissue surface (increasing the tissue depth). The bottom-row pictures show that many proximal tibial growth plate chondrocytes incorporated EdU in 2 h. These actively proliferating cells were all located in the upper half of the columnar zone (CZ). The labeling of cells in all tissue layers, including the hypertrophic zone (HZ), at day 2 and the loss of this labeling by day 7 indicate that columnar chondrocytes continued to divide and that their progeny progressed through all stages of cell maturation within a few days. By day 12, EdU-labeled cells could only be detected in the resting zone (RZ) of the growth plate. These slowly cycling cells were likely growth plate chondrocyte stem cells. Note that the primary and secondary ossification centers (POC and SOC, respectively) feature bone marrow and bone cells that massively incorporated EdU within 2 h, but that only a few cells were still labeled by day 12, indicating an abundance of actively proliferating cells and a low proportion of slowly dividing cells, presumably corresponding to hematopoietic and skeletal stem cells