## 4 Discussion
Our screening assay also allows for further examination of specific combinations and single growth factor addition benefits or otherwise. For example, we found that the addition of either TGF-\(\beta\)1 or TGF-\(\beta\)2 to GDF6/Wnt3a resulted in similar alcian blue production rates and subsequently did not appear to enhance NP marker expressions either. This suggested that TGF-\(\beta\)1 or TGF-\(\beta\)2 have no added value to the GDF6/Wnt3a combination. However, subsequent pellet cultures revealed that GDF6/Wnt3a with TGF-\(\beta\)1 resulted in significantly higher CD24 and COL2A1 expression levels, and showed a trend of enhanced ACAN and SOX9 expression levels. These observations further highlighting the context-dependent nature of cell culture and the effects of growth factors on the respective cells. Notwithstanding, our results require some consideration before interpretation. One limitation of our study is the limited number of donor samples applied, particularly for the initial screening assay. Due to the high cost of applied growth factors and relatively long culture period, the initial screening of 86 different combinations was assessed on one single-sourced MSC. Moreover, the initial alcian blue screening was performed without correction for cell numbers or DNA content. Differences could in theory be caused by differences in cell content. However, we believe this is unlikely, as the long (3 weeks) culture period led all conditions to a confluent culture condition, and the differences at the time of assessment were likely minimal. Another study limitation to consider is that we did not assess outcomes of our monolayer and pellet cultures at different time points. Such particular combinations might have revealed more beneficial outcomes at shorter or longer stimulation periods. This requires further examination. Overall, a need exists for novel regenerative strategies to treat intervertebral disc diseases. Currently, cell therapy [26] and growth factor injection [17] is gaining momentum, however, further validation and optimization of different regenerative products will likely be needed [64]. One potential approach for enhancing cellular products is priming the cells before transplantation [65], to promote an optimal anabolic state, such that the transplanted cells have the highest potential to contribute to the reorganization of the disc matrix or otherwise direct regional cells to do so [30]. Growth factors could be a potent tool in this regard. However, caution is required. Growth factors often function in context-dependent manners, and as such not every MSC population sourced from different donors might respond similarly. For clinical application and marketability, cell-product quality control is key, and this aspect should be taken into consideration [3; 4; 66]. Moreover, growth factors are generally quite expensive, particularly when applied over longer culture periods and for large-scale production. Thus, their application could hinder commercialization, by potentially increasing the price of cellular therapy production. Again, care should be taken that the benefits of growth factor stimulation outweigh the additional expense of the cellular product. From our screening of 86 different growth factor combinations for MSC to NP cell differentiation, we found that Wnt3a could potentially be an impactful factor when applied in 2D monolayer, although the mechanisms and requirements for these effects compel further exploration. Of all combinations analyzed, only the combination of GDF5 and TGF-\(\beta\)1 was able to result in fully chondrogenic pellets when applied in 3D cultures, and it might be the most potent combination for MSC to NP cell differentiation. D.S. and J.S. ; conceptualization, T.N.E.V., K.M., J.S. ; methodology, T.N.E.V., K.M., J.S. ; formal analysis, D.S., M.S., M.W. ; resources, J.S. ; writing--original draft preparation, J.S., T.N.E.V., K.M., D.S., M.S., M.W. ; writing--review and editing, J.S. ; visualization, D.S., M.S., M.W. ; supervision, D.S., M.S., M.W. ; funding acquisition.