The effects of 3D culture on the expansion and maintenance of nucleus pulposus progenitor cell multipotency

Abstract

Introduction: Low back pain (LBP) is a global health concern. Increasing evidence implicates intervertebral disk (IVD) degeneration as a major contributor. In this respect, tissue-specific progenitors may play a crucial role in tissue regeneration, as these cells are perfectly adapted to their niche. Recently, a novel progenitor cell population was described in the nucleus pulposus (NP) that is positive for Tie2 marker. These cells have self-renewal capacity and in vitro multipotency potential. However, extremely low numbers of the NP progenitors limit the feasibility of cell therapy strategies. Objective: Here, we studied the influence of the culture method and of the microenvironment on the proliferation rate and the differentiation potential of human NP progenitors in vitro. Method: Cells were obtained from human NP tissue from trauma patients. Briefly, the NP tissue cells were cultured in two-dimensional (2D) (monolayer) or threedimensional (3D) (alginate beads) conditions. After 1 week, cells from 2D or 3D culture were expanded on fibronectin-coated flasks. Subsequently, expanded NP cells were then characterized by cytometry and tri-lineage differentiation, which was analyzed by qPCR and histology. Moreover, experiments using Tie2 + and Tie2 − NP cells were also performed. Results: The present study aims to demonstrate that 3D expansion of NP cells better preserves the Tie2 + cell populations and increases the chondrogenic and osteogenic differentiation potential compared to 2D expansion. Moreover, the cell sorting experiments reveal that only Tie2 + cells were able to maintain the pluripotent gene expression if cultured in 3D within alginate beads. Therefore, our results highly suggest that the maintenance of the cell's multipotency is mainly, but not exclusively, due to the higher presence of Tie2 + cells due to 3D culture. Conclusion: This project not only might have a scientific impact by evaluating the influence of a two-step expansion protocol on the functionality of NP progenitors, but it could also lead to an innovative clinical approach.

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

“…For example, FGF2-stimulated WTC culture increased the final Tie2 + fraction 7.5-fold compared with standard PCC. Comparatively, work by Guerrero et al [ 26 ] revealed a 1.8-fold increase in Tie2 rates, comparing their human NP cells cultured in alginate bead versus a standard monolayer culture. A 3.6-fold increase was reported by Zhang et al [ 30 ], who employed spheroid-based suspension cultures.…”

“…These Tie2 + cells were characterized as undifferentiated cells with multipotency and possessing high self-renewal abilities [ 22 , 23 , 24 , 25 ]. Therefore, we and others have proposed Tie2 + NPPC as a potent cell source for regenerative cell therapies against IVD degeneration [ 26 ]. However, their utilization is hindered by low Tie2-expressing cell yields from NP tissue, in particular from commonly available older and degenerated tissue sources [ 21 , 25 ].…”

“…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.…”

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.…”

“…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.…”