Gli1-expressing stromal cells are highly reparative precursors of long-lived chondroprogenitors in the fetal murine limb

Single-cell RNA sequencing of cells dissociated from Hindlimb at E14.5 embryos to identify how catch-up growth happens during injury and to show transcriptional identities of contributors to chondrocytes in p21-overexpression induced injury model. Female Gli1CreER/CreER mouse was crossed with Col2a1-rtTA; TigreTRE-p21/TRE-p21; R26LSL-tdT/LSL-tdT male. At E12.5, the pregnant mouse was administered doxycycline in water (1 mg/ml with 0.5% sucrose) and food, while Tamoxifen was orally gavaged (120 μg/g) at E13.5. At E14.5, the hindlimb samples (femur and tibia) were carefully dissected. Following, the tissue underwent treatment with Accumax in a rocker at 31°C for 25 minutes and they were gently shaken every 10 min. The supernatant was carefully transferred into a tube and placed on ice for preservation, while the cartilaginous portions were exposed to Collagenase B (5 mg/ml; Sigma) for ~90 min at 37°C in a water bath (and pipetted every 20 min). To stop collagenase activity, we then added 700 µl of cold DMEM+20% FBS for each tube. We then incorporated outer and inner supernatants, and the samples were resuspended in 20 volumes of 1× RBC lysis buffer and incubated for 10 minutes at room temperature on a rocker. As a final step, the cell suspension was filtered through 40-μm Flowmi® Cell Strainers and cells were stained with DAPI (stock concentration: 0.05 µg/ml) and transferred into Falcon™ Round-Bottom Polypropylene (Fisher Scientific). The tdT+ and tdT- cells were sorted using flow cytometry and the samples were then multiplexed based on sex and conditions.