Are olfactory ensheathing cells a promising cell therapy tool?

Olfactory Ensheathing Cells (OECs) show a peculiar plasticity and represent a unique population in the olfactory system supporting the continuous neuronal turnover and sheathing olfactory axons. They exhibit antigenic and morphological characteristics both of astrocytes and of Schwann Cells. In vitro, OECs promote axonal growth, moreover in vivo they can form myelin, promoting remyelination of damaged axons. In the last two decades, OECs have emerged as possible supportive cells for regeneration and functional recovery of damaged Central Nervous System (CNS). A characterization was performed both by flow cytometry and immunocytochemistry for the following markers: Vimentin, S-100β, Nestin, Glial Fibrillary Acidic Protein, Myelin, Neural Cell Adhesion Molecule, Low-affinity Nerve Growth Factor Receptor p75, Microtubule Associated Protein-2 and Protein Gene Product 9.5. In order to study the modulation of these markers, OECs were also grown in different culture conditions: standard or serum-free media with/without Growth Factors (GFs), such as basic Fibroblast Growth Factor and Glial Derived Neurotrophic Factor. Basal apoptosis was evaluated by annexin and propidium iodide analysis as well as after exposition to 6-hydroxydopamine (6-OHDA). Neural stem cells and a neuroblastoma cell line (SH-SY5Y) were used as control, primary OECs were prepared from postnatal mouse (P1) olfactory bulbs. Moreover, neuroprotective properties of OECs on 6-OHDA-treated cells were evaluated by an in vitro co-culture system or addition of OEC conditioned medium. We observed: 1) change of OEC usual morphology, reduction of both cell viability and marker expression in serum-free medium; 2) positive influence of GFs on both viability and marker expression; 3) no increased apoptosis after a prolonged exposition to 6-OHDA; 4) OEC neuroprotective effect, albeit non statistically significant, on 6-OHDA treated SH-SY5Y cells. These peculiar properties of OECs might render them as useful potential clinical agents being able to support injured CNS

Longitudinal Tracking of Human Fetal Cells Labeled with Super Paramagnetic Iron Oxide Nanoparticles in the Brain of Mice with Motor Neuron Disease

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival

High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

Intra-cellular ferric ions measurement.

<p>Cells were incubated for 24, 48 or 72 hours with 35 µg/ml of SPIOn. Iron content expressed as Fe and Fe₃O₄ in control-samples of AFC, CVC and physiological solution along with a Fe₃O₄ standard at 35 ppm are reported. Unlabeled cells represent control condition (CTR). Data (mg/L) are expressed as mean ± SD and analyzed with two way analysis of variance (ANOVA).</p

Role of ongoing degeneration on cell migration.

<p><b>a</b> 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>b</b> hAFCs and <b>c</b> 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. <b>d</b> timeline of migration experiments; <b>e</b> hAFCs and <b>f</b> 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.</p

Evaluation of SPIOn labeled cell biological properties and migration.

<p><b>a</b> Proliferative and <b>b</b> metabolic rates of hCVCs appeared not affected by SPIOn presence, as previously demonstrated by our group for hAFCs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078435#pone.0078435-Bigini1" target="_blank">[20]</a>; <b>c</b> 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.</p

Flow cytometric analysis.

<p>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 (<b>a,b</b>): when they were mixed, a single cell population was detected (<b>c</b>); different labeling of hCVCs (<b>d,g</b>) and hAFCs (<b>e,h</b>), before mixing the cells, allowed us to identify simultaneously both the cell population confirming their comparable size and complexity (<b>f</b>).</p

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

<p><b>a</b> hAFCs and <b>b</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; <b>c</b> hAFCs and <b>d</b> 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.</p

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

<p><b>a</b> 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>b</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.</p

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

<p><b>a</b> hAFCs and <b>b</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); <b>c</b> hAFCs and <b>d</b> 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 <b>e</b> 6, <b>f</b> 12, <b>g</b> 24, <b>h</b> 48 and <b>i</b> 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.</p