Spheroid-Like Cultures for Expanding Angiopoietin Receptor-1 (aka. Tie2) Positive Cells from the Human Intervertebral Disc

Lower back pain is a leading cause of disability worldwide. The recovery of nucleus pulposus (NP) progenitor cells (NPPCs) from the intervertebral disc (IVD) holds high promise for future cell therapy. NPPCs are positive for the angiopoietin-1 receptor (Tie2) and possess stemness capacity. However, the limited Tie2+ NPC yield has been a challenge for their use in cell-based therapy for regenerative medicine. In this study, we attempted to expand NPPCs from the whole NP cell population by spheroid-formation assay. Flow cytometry was used to quantify the percentage of NPPCs with Tie2-antibody in human primary NP cells (NPCs). Cell proliferation was assessed using the population doublings level (PDL) measurement. Synthesis and presence of extracellular matrix (ECM) from NPC spheroids were confirmed by quantitative Polymerase Chain Reaction (qPCR), immunostaining, and microscopy. Compared with monolayer, the spheroid-formation assay enriched the percentage of Tie2+ in NPCs’ population from ~10% to ~36%. Moreover, the spheroid-formation assay also inhibited the proliferation of the Tie2- NPCs with nearly no PDL. After one additional passage (P) using the spheroid-formation assay, NPC spheroids presented a Tie2+ percentage even further by ~10% in the NPC population. Our study concludes that the use of a spheroid culture system could be successfully applied to the culture and expansion of tissue-specific progenitors.Financial support was received from iPSpine H2020 project under the grant agreement #825925, the Swiss National Science Foundation Project 310030E_192674/1, both to B.G., and from a fellowship by the China Scholarship Council to X.Z. Further funds were received from the Swiss Society of Orthopaedics (SGOT), the clinical trials unit of Bern University Hospital, and by an Eurospine Task Force Research grant #2019_22 to C.E.A