Deferoxamine Preconditioning of Neural-Like Cells Derived from Human Wharton's Jelly Mesenchymal Stem Cells as a Strategy to Promote Their Tolerance and Therapeutic Potential: An In Vitro Study

Abstract

Transplantation of neural-like cells is considered as a promising therapeutic strategy developed for neurodegenerative disease in particular for ischemic stroke. Since cell survival is a major concern following cell implantation, a number of studies have underlined the protective effects of preconditioning with hypoxia or hypoxia mimetic pharmacological agents such as deferoxamine (DFO), induced by activation of hypoxia inducible factor-1 (HIF-1) and its target genes. The present study has investigated the effects of DFO preconditioning on some factors involved in cell survival, angiogenesis, and neurogenesis of neural-like cells derived from human Wharton's jelly mesenchymal stem cells (HWJ-MSCs) in presence of hydrogen peroxide (H2O2). HWJ-MSCs were differentiated toward neural-like cells for 14 days and neural cell markers were identified using immunocytochemistry. HWJ-MSC-derived neural-like cells were then treated with 100 µM DFO, as a known hypoxia mimetic agent for 48 h. mRNA and protein expression of HIF-1 target genes including brain-derived neurotrophic factors (BDNF) and vascular endothelial growth factor (VEGF) significantly increased using RT-PCR and Western blotting which were reversed by HIF-1α inhibitor, while, gene expression of Akt-1, Bcl-2, and Bax did not change significantly but pAkt-1 was up-regulated as compared to poor DFO group. However, addition of H2O2 to DFO-treated cells resulted in higher resistance to H2O2-induced cell death. Western blotting analysis also showed significant up-regulation of HIF-1α, BDNF, VEGF, and pAkt-1, and decrease of Bax/Bcl-2 ratio as compared to poor DFO. These results may suggest that DFO preconditioning of HWJ-MSC-derived neural-like cells improves their tolerance and therapeutic potential and might be considered as a valuable strategy to improve cell therapy.

Keywords: BDNF; Deferoxamine; Neural-like cells; Preconditioning; VEGF.

Fig. 1

Flow cytometry analysis of HWJ-MSCs. The black histograms are the antibody-stained HWJ-MSCs and the red histograms are the isotype control cells. The proportion of stained cells was provided in the histogram for each marker. HWJ-MSCs expressed mesenchymal markers CD44, CD73, CD90, and CD105 and did not express hematopoietic cell markers CD34 and CD45 (Color figure online)

Fig. 2

Neural differentiation, Cresyl Violet staining, and immunocytochemistry staining on differentiated HWJ-MSCs to neural-like cells. a Cytoplasm in spindle-like shape HWJ-MSCs before neural differentiation, retracted toward the nucleus, and shaped to contracted multipolar cell body with some process-like extensions. Arrows show neural-like cells with branching processes. HWJ-MSCs differentiated to neural-like cells with pyramidal or spherical forms and neurite-like extensions on day 7 and b day 14 (×20 magnification). c HWJ-MSCs differentiated to neural-like cells after staining with cresyl violet. Dark blue particles in the cytoplasm show Nissl bodies (×20 magnification). In immunocytochemistry, cells with brown color exhibit the neural markers d nestin, e β-tubulin III, f GFAP, g NF, and h CNPase in differentiated HWJ-MSCs. Scale bars 50 µm (Color figure online)

Fig. 3

Total cell viability and neural-like cell percentage after preconditioning with DFO. The cells were treated with different concentrations of DFO for 24, 48, and 72 h. a Viability of total cells was measured by trypan blue exclusion test. b Percentage of neural-like cells was measured by cresyl violet staining. c Percentage of the neural-like cells was also evaluated by nestin immunoreactivity. Groups that treated with 0 μM DFO were regarded as control in all tests, results were reported as the mean ± SEM (n = 4), and were significant at *P < 0.05, **P < 0.01, and ***P < 0.001 compared to control

Fig. 4

Total cell viability and neural-like cell percentage of preconditioned cells with DFO after exposure to hydrogen peroxide. The cells were treated with 100 µM DFO for 48 h, then exposed to 500 µM H₂O₂ for 24 h. a Cell viability after exposure to H₂O₂ was measured by trypan blue exclusion test. b Percentage of neural-like cells after exposure to H₂O₂was measured by Nissl body staining using cresyl violet. c Percentage of the neural-like cells was also evaluated by nestin immunoreactivity. Untreated cells are not treated with DFO and not with H₂O₂. Results are reported as the mean ± SEM (n = 4) and are significant compared to untreated cells (***P < 0.001) and compared to treated cells only with H₂O₂ (^### P < 0.001)

Fig. 5

DFO preconditioning induced gene regulation. a Semiquantitative analysis of genes expression of BDNF, VEGF, Bcl-2, Bax, and Akt-1 was performed by densitometry. The expressions of genes are normalized to optical β-Actin density. Data were represented as mean ± SEM and were significant at ***P < 0.001 versus untreated cells while at ^### P < 0.001 versus treated cells with DFO and HIF-1α inhibitor (n = 4). b BDNF and VEGF mRNA levels in the cells were up-regulated after 48 h treatment with 100 μM DFO, revealed by RT-PCR measurements. No significant change in the expression of the proapoptotic gene, Bax and anti-apoptotic genes, Bcl-2 and Akt-1 was observed under 100 μM DFO for 48 h. The gene expression pattern of BDNF and VEGF displayed a shift to the control with pretreatment by HIF-1α inhibitor for 4 h. β-Actin was used as a loading control

Fig. 6

DFO preconditioning induced protein regulation. a Quantitative analysis of protein expression of HIF-1α, BDNF, VEGF, Bcl-2, Bax, pAkt-1, and Akt-1 was performed by densitometry. The expressions of proteins were normalized to optical β-Actin density. Data showed that DFO markedly elevates HIF-1α, BDNF, VEGF, and pAkt-1 protein levels, while were reversed by HIF-1α-I pretreatment. Data were represented as mean ± SEM and were significant at ***P < 0.001 versus untreated cells, while at ^# P < 0.05 and ^### P < 0.001 versus treated cells with DFO and HIF-1α inhibitor (n = 4). b Western blot analysis showed significant up-regulation of HIF-1α, BDNF, VEGF, and pAkt-1 after 48 h treatment with 100 µM DFO. The protein levels of HIF-1α, BDNF, VEGF, and pAkt-1 induced by the DFO treatment, down-regulated by pretreatment with HIF-1α inhibitor (HIF-1α-I). The HIF-1 activation was inhibited by the HIF-1α inhibitor. β-Actin was used as a loading control

Fig. 7

Protein levels of DFO preconditioned cells after the termination of H₂O₂ treatment. a Quantitative analysis of protein expression of HIF-1α, BDNF, VEGF, Bcl-2, Bax, pAkt-1, and Akt-1 was performed by densitometry after H₂O₂ treatment in the cells with preconditioning with DFO. The expressions of proteins were normalized to optical β-Actin density. b The protective proteins Bcl-2 and pAkt-1 decreased and proapoptotic protein Bax increased significantly after H₂O₂ treatment in the cells without preconditioning with DFO. c The Bax/Bcl-2 ratio showed significant up-regulation in group unpretreated with DFO before exposure to H₂O₂ compared to treated groups with DFO. Data were represented as mean ± SEM and were significant at **P < 0.01 and ***P < 0.001 versus untreated cells and were significant at ^## P < 0.01 and ^### P < 0.001 versus treated cells only with H₂O₂ (n = 4)