Tagln+ progenitors contribute to the development and maintenance of nucleus pulposus

Failure of intervertebral disc components, e.g. the nucleus pulposus causes intervertebral disc disease and associated low-back pain. Despite the high prevalence of disc disease, the changes in intervertebral disc cells and their regenerative potential with ageing and degeneration are not fully elucidated. Understanding the cell lineage, cell differentiation and maintenance of nucleus pulposus may have therapeutic application for the regeneration of degenerative disc, with significant impact for healthy ageing. Here we found that TAGLN expressing cells are present in human healthy nucleus pulposus, but diminish in degenerative disc. By lineage analyses in mice, we found cells in the nucleus pulposus are derived from a peripherally located population of notochord-derived Tagln expressing cells (PeriNP cells). The PeriNP cells are proliferative and can differentiate into the inner part of the nucleus pulposus. The Tagln+ cells and descendants diminish during aging and puncture induced disc degeneration. The maintenance and differentiation of PeriNP cells is partially regulated by Smad4 dependent signaling. Removal of Smad4 by nucleus pulposus specific Cre (Foxa2mNE-Cre), results in decreased Tagln+ cells and abnormal disc morphology, leading to disc degeneration. Our findings propose that the PeriNP Tagln expressing cells are a pool of notochord-derived progenitors that are important for maintenance of the nucleus pulposus and provide insights for regenerative therapy against intervertebral disc degeneration. The Tagln-CreERt2;R26tdTomato mice were activated with tamoxifen at P5 stage, and IVDs from lumbar region (L1 to L6 levels) and tail region (proximal T5 to T9 levels) were harvested at P10 and 8-week stages. Muscles and blood vessels were removed as much as possible and blood cells were harshly washed away with HBSS (Gibco). Remaining IVD tissues were cut into small fragments and digested with 0.25% Dispase (Worthington) and 0.25% type II collagenase (Worthington) in HBSS (5ml in total) at 37ºC with gentle shaking for 1 hour. Digestion was filtered with a 40µm cell strainer. The cells washed with HBSS twice and sent for sequencing using Chromium single cell platform (10X Inc.) at the Centre for PanorOmic Sciences (CPOS, HKU).