Single-cell profiling of the nucleus pulposus (NP) supports the Shh+ cells as NP stem cells

The physiological function of the intervertebral disc (IVD) depends on the cellular and molecular composition of the nucleus pulposus (NP), which plays a key role in managing the mechanical loading of the IVD.We conducted single-cell RNA sequencing (scRNA-seq) analysis to better characterize the NP cells, particularly the Shh-tdTom+ NP cells, in the adult Shh-CreER; Rosa26tdTomato mice. After five consecutive Tamoxifen injections to the 1-month-old Shh-CreER; Rosa26tdTomato mice, lumbar and coccygeal IVDs were harvested 2 weeks later. CD45+ immune cells were removed by Fluorescence-Activated Cell Sorting (FACS) to focus on the non-immune cells in the IVD. The digested IVD cells were then subjected to 10X Genomics scRNA-seq.