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

Abstract

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.

Towards Tissue-Specific Stem Cell Therapy for the Intervertebral Disc: PPARδ Agonist Increases the Yield of Human Nucleus Pulposus Progenitor Cells in Expansion

“…The classic and common culture conditions are based on 2D culture and normoxic environment [13][14][15]. In this research, we primarily wanted to compare the impact of chemical treatments on the Tie2+ NPPCs population [16,17]. For this reason, the cells were cultured within a normoxic environment.…”

“…Cells' morphology was quantified by using ImageJ v1.53c software on the microscopic pictures, using "cell body circularity" functionality [28,29]. An example of how the cells' outlines were defined by manual segmentation can be found in a previous study [17].…”

“…Therefore, new protocols for (i) how to maintain the number of initially cell-sorted NPPCs in cell culture and (ii) the protection of their stemness are urgently warranted. Recent work could clearly demonstrate that changing the expansion protocol from 2D to 3D can indeed increase the Tie2+ cell's yield [16,17]. Moreover, NPPC protection through molecular injection could rebuild the cell's homeostasis in IVDD; it could evolve as a new approach for IVDD therapy.…”

Effect of Whole Tissue Culture and Basic Fibroblast Growth Factor on Maintenance of Tie2 Molecule Expression in Human Nucleus Pulposus Cells

“…As such, a need exists to optimize or develop new culture methods that enable the maintenance of Tie2-expressing NPPC. A variety of studies have investigated different approaches to improve culture conditions [ 26 , 29 , 30 , 31 , 32 ] or isolation methods [ 22 ] to increase the number of Tie2-expressing cells. Bovine-derived NP cell studies have suggested a synergistic beneficial role of fibroblast growth factor 2 (FGF2; also known as basic fibroblast growth factor) and a hypoxic culture environment.…”

“…For example, the Tie2 + NPPC was originally discovered by their formation of spheroid colony-forming units (CFUs) within a methylcellulose semi-solid medium [ 21 ]. Moreover, Zhang et al reported a high colony formation rate and Tie2 positivity rate by using an ultra-low attachment T75 flask [ 30 ]. Similarly, Guerrero et al recently found enhanced Tie2 + NPPC maintenance by employing a 3D environment of alginate-beads with enhanced pluripotency markers.…”