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Mesenchymal stromal cells (MSCs) possess immunomodulatory abilities that make them a promising candidate for the treatment of immune-mediated diseases, particularly those with an autoimmune component. We have recently shown that preconditioning human bone marrow (BM) MSCs with Vadadustat – hypoxia mimicking agent, may constitute a valuable approach to improve the therapeutic properties of MSCs. Vadadustat is one of hypoxia inducible factor prolyl hydroxylase (HIF-PH) inhibitors, known also as HIF stabilizers. MSCs isolated from Wharton’s jelly (WJ) constitute an attractive alternative to BM-MSCs. Perinatal tissues are available in unlimited amounts, and WJ-MSCs have a greater proliferative activity than BM-MSC with comparable immunomodulatory potential. Therefore, the objective of this study was to compare the effect of HIF-PH inhibitor on these two cell types. Moreover, we aimed to evaluate if HIF stabilizers affects the interaction between MSCs and lymphocytes in terms of T regulatory (Tregs) cells formation. First, we have assessed the effect of two different HIF-PH inhibitors on the HIF1α expression. Western blot analysis revealed that IOX4 (selective HIF prolyl-hydroxylase 2 inhibitor) used in concentration 5 µM caused substantially stronger HIF1α stabilization in both BM-MSCs and WJ-MSCs than Vadadustat in concentration 40 µM. Moreover, the effect induced by IOX4 lasted at least 48h whereas the effect of Vadadustat was not longer than 24h. The effect of IOX4 on viability and proliferative activity of BM-MSCs and WJ-MSCs was assessed using MTT assay and BrdU assay, respectively. In both tests WJ-MSCs appeared to be significantly more sensitive to IOX4 treatment than BM-MSCs. In comparison to control (solvent only), the mean viability of BM-MSCs and WJ-MSCs after 48h treatment with IOX4 decreased by 5% and 18%, respectively (the difference in response between two cell types – p=0.02). Analogous decrease in BrdU assay amounted 5% and 52% for BM-MSCs and WJ-MSCs, respectively (the difference in response between two cell types – p=0.03). Next we evaluated the effect of both analyzed MSC types on Treg formation in the population of allo-activated lymphocytes. Both BM-MSCs and WJ-MSCs significantly increased the proportion of Tregs (defined as CD4+CD25++CD127-) in CD3+CD4+ cells after 3 days of co-culture of allo-activated lymphocytes with MSCs. An average increase in Tregs proportion in the presence of BM-MSCs and WJ-MSCs amounted 63% and 43%, respectively (p<0.05 for both MSC types in comparison to allo-activated lymphocytes without MSCs). Pretreatment of MSC with IOX4 before co-culture with lymphocytes did not significantly affect the effects of MSCs on Tregs formation regardless of MSC type used.
To summarize, IOX4, selective HIF prolyl hydroxylase 2 inhibitor has cytotoxic effect on hWJ-MSCs, which appeared to be significantly stronger than on hBM-MSCs. This difference in response may be due to the various physiological conditions of the niches from which both cell types are derived. These results suggest that mimicking hypoxia may have different effects on MSCs from various sources. Nevertheless, our results indicate that these differences do not necessarily translate into the effects of complex interactions of MSCs with immune cells.
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