Dose dependent side effect of superparamagnetic iron oxide nanoparticle labeling on cell motility in two fetal stem cell populations

Abstract

Multipotent stem cells (SCs) could substitute damaged cells and also rescue degeneration through the secretion of trophic factors able to activate the endogenous SC compartment. Therefore, fetal SCs, characterized by high proliferation rate and devoid of ethical concern, appear promising candidate, particularly for the treatment of neurodegenerative diseases. Super Paramagnetic Iron Oxide nanoparticles (SPIOn), routinely used for pre-clinical cell imaging and already approved for clinical practice, allow tracking of transplanted SCs and characterization of their fate within the host tissue, when combined with Magnetic Resonance Imaging (MRI). In this work we investigated how SPIOn could influence cell migration after internalization in two fetal SC populations: human amniotic fluid and chorial villi SCs were labeled with SPIOn and their motility was evaluated. We found that SPIOn loading significantly reduced SC movements without increasing production of Reactive Oxygen Species (ROS). Moreover, motility impairment was directly proportional to the amount of loaded SPIOn while a chemoattractant-induced recovery was obtained by increasing serum levels. Interestingly, the migration rate of SPIOn labeled cells was also significantly influenced by a degenerative surrounding. In conclusion, this work highlights how SPIOn labeling affects SC motility in vitro in a dose-dependent manner, shedding the light on an important parameter for the creation of clinical protocols. Establishment of an optimal SPIOn dose that enables both a good visualization of grafted cells by MRI and the physiological migration rate is a main step in order to maximize the effects of SC therapy in both animal models of neurodegeneration and clinical studies.

Figure 1. Evaluation of SPIOn labeled cell biological properties and migration.

a Proliferative and b metabolic rates of hCVCs appeared not affected by SPIOn presence, as previously demonstrated by our group for hAFCs ; c hAFCs and hCVCs were incubated for 72 hours with SPIOn (35 µg/ml) and then migration assay was performed. Data are expressed as a percentage of migrated cells. ***p<0.001 versus respective (unlabeled, control cells, CTR); °°p<0.01 versus SPIOn labeled hCVCs. One way analysis of variance (ANOVA) as specified in the statistical section.

Figure 2. Evaluation of intra-cellular ROS during internalization and subsequent removal of SPIOn.

a hAFCs and b hCVCs were plated and labeled with DAF-FM to visualize intra-cellular ROS, before being incubated with SPIOn (35 or 100 µg/ml). ROS measurement was performed every hour (from 1^st to 5^th) during SPIOn internalization (see experimental scheme). Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). No statistically significant differences were observed between CTR and SPIOn labeled cells. ***p<0.001 versus CTR; °°°p<0.001 versus SPIOn (35 µg/ml). Two way analysis of variance (ANOVA); c hAFCs and d hCVCs were plated and labeled with DAF-FM and then were incubated with SPIOn (35 µg/ml). ROS evaluation was done at different time (6, 12, 24, 48 and 72 hours) after SPIOn addition. Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). ***p<0.001 versus respective 6 hours; °°°p<0.001 respective 12 hours; +++p<0.001 versus respective 24 hours. Two way analysis of variance (ANOVA) as specified in the Statistical section. To better visualize the significant difference in intra-cellular ROS formation between hAFCs and hCVCs, we compared the two cellular groups at e 6, f 12, g 24, h 48 and i 72 hours after SPIOn removal. Data are expressed as ROS fluorescent signal normalized for Digitonin-PI. Unlabeled cells represent control condition (CTR). **p<0.01 and ***p<0.001 versus hAFCs. Two way analysis of variance (ANOVA), as specified in the statistical section.

Figure 3. Migration and survival of hAFCs and hCVCs labeled with different SPIOn concentrations.

a Cells were labeled with different concentration of SPIOn for 72 hours before migration assay. Data are expressed as a percentage of migrated cells. *p<0.05, **p<0.01 and ***p<0.001 versus respective CTR (control, unlabeled cells). Two way analysis of variance (ANOVA); as specified in the statistical section; b hAFCs and hCVCs labeled with different SPIOn concentrations were used to test SPIOn toxicity by MTS assay. Data are expressed as a percentage of viable cells versus unlabeled cells used as control (CTR). Two way analysis of variance (ANOVA); as specified in the statistical section.

Figure 4. Flow cytometric analysis.

Fluorescent cell linker labeled hCVCs (here PKH26) and hAFCs (here PKH67) were analyzed by flow cytometry in order to investigate differences in size and cell complexity. Representative figure of unlabeled hCVCs and hAFCs (a,b): when they were mixed, a single cell population was detected (c); different labeling of hCVCs (d,g) and hAFCs (e,h), before mixing the cells, allowed us to identify simultaneously both the cell population confirming their comparable size and complexity (f).

Figure 5. Effect of serum concentration on the migration of SPIOn labeled hAFCs and hCVCs.

a hAFCs and b hCVCs were incubated for 72 hours with different concentration of SPIOn and then migration assay was performed. In these experimental setting, medium with 10% FBS was added in the bottom chamber. Data are expressed as a percentage of migrated cells. Unlabeled cells represent control condition (CTR). **p<0.01 and ***p<0.001 versus serum free medium. Two way analysis of variance (ANOVA) as specified in the Statistical section; c hAFCs and d hCVCs were labeled with SPIOn 35 µg/ml (72 hours of incubation) and migration assay was performed. In these experiments medium plus 10, 20 or 30% FBS was added in the bottom chamber. Data are expressed as a percentage of migrated cells. Unlabeled cells represent control condition (CTR). *p<0.05 and ***p<0.001 versus serum free medium. °p<0.05 and °°°p<0.001 versus 10% FBS; +p<0.05 and +++p<0.001 versus 20% FBS. Two way analysis of variance (ANOVA) as specified in the statistical section.

Figure 6. Role of ongoing degeneration on cell migration.

a SH-SY5Y cells were plated and treated with 6-OHDA 100 µM. After 1, 4 and 6 hours viability was measured by MTS assay. Data are expressed as a percentage of viable cells versus CTR (no 6-OHDA treated cells). ***p<0.001 versus CTR. One way analysis of variance (ANOVA) as specified in the Statistical section; b hAFCs and c hCVCs were incubated for 72 hours with SPIOn (35 µg/ml) and then were treated with 6-OHDA (100 µM). MTS assay was performed 1, 4 and 6 hours after 6-OHDA/toxin addition. Data are expressed as a percentage of viable cells versus respective CTR (no 6-OHDA treated cells). Two way analysis of variance (ANOVA) as specified in the Statistical section. d timeline of migration experiments; e hAFCs and f hCVCs were labeled with SPIOn 35 µg/ml (72 hours of incubation) followed by the migration assay. In these experiments, SH-SY5Y cells were plated in the bottom chamber and then treated with/exposed to 6-OHDA (100 µM). Migration was evaluated 1, 4 and 6 hours after treatment. Data are expressed as a percentage of migrated cells. Unlabeled cells represent control condition (CTR). ***p<0.001 versus not treated (NT) SH-SY5Y cells; °p<0.05, °°p<0.01 and °°°p<0.001 versus 1 hour treatment; +++p<0.001 versus 4 hours treatment. Two way analysis of variance (ANOVA) as specified in the statistical section.