De-differentiation study of porcine notochordal cells
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Introduction: Intervertebral disc (IVD) degeneration is a worldwide health problem, and causes low back-pain in humans and dogs. New regenerative treatments are necessary, because current treatments are invasive, expensive,, and only focus on end-stage disease. Notochordal cells (NCs) derive from the notochord and have shown to replenish and instruct the IVD cell population, acting as a tissue specific progenitor-cell, and are considered to have anti-inflammatory and regenerative capacities. Because of all the challenges with regard to collecting, expanding, and culturing NCs, an alternative strategy is desirable. A promising approach could be the use of induced pluripotent stem cells (iPSCs). However, to optimize the differentiation of iPSCs to a notochordal-like cell type (NLC), the complete (epi)genetic landscape of NCs should be delineated first, by using native NCs and de-differentiated NCs as comparators. Aim: This study aimed at generating and validating a culture system in which porcine NCs lose their specific phenotype in a controlled manner. When succeeded, these de-differentiated NCs can be used as comparators in follow up (epi)genetic analysis to delineate which specific (epi)genetic characteristics are lost and therefore typical for native NCs. In the end, this will contribute to successfully generate iPSC lines with an optimal capacity to differentiate towards mature iPS-NLCs. Methods. Porcine NCs were subjected to different culture conditions including low glucose levels (0.006 M), high glucose levels (0.1 M), and the addition of fetal calf serum (FCS) in both a 2D (monolayer) and 3D (alginate bead) culture system. The NC morphology and phenotype, extra-cellular matrix production, and cell proliferation were determined by gene expression profiling, histological evaluation and measuring the DNA and glycosaminoglycan (GAG) content of the cultures. Results: No distinct adverse effect of the high glucose conditions both with and without FCS was encountered with regard to the expression of different NC markers, apoptotic, and extra-cellular matrix associated genes compared to the low glucose conditions in both 2D and 3D culture systems. The DNA and GAG content of the monolayers were slightly decreased in the high glucose conditions and with the addition of FCS compared with the low glucose conditions. The DNA and GAG content of the alginate beads were increased in the high glucose conditions, whereas these were slightly decreased by the addition of FCS compared with the low glucose conditions. The cells cultured with FCS showed predominantly a spindle-shaped phenotype and a loss of vacuoles both in the monolayers as well as in the alginate beads. Conclusions: The results of the current study suggest that FCS induces de-differentiation of NCs in both a 2D and 3D culture system, in contrast to increasing the glucose concentration. However, since the composition of FCS is uncertain and batches can vary, future research should delineate which specific growth factor can be used to induce a more controlled de-differentiation. Furthermore, because of the limitations of this pilot study, it is yet infeasible to draw definite conclusions with regard to the optimal de-differentiation culture set-up and therefore follow up studies are necessary.