Stem cell therapy: how to do it right

Peer reviewe

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

Amyotrophic lateral sclerosis: applications of stem cells – an update

Lidia Cova1, Vincenzo Silani21Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milano, Italy; 2Department of Neurology and Laboratory of Neuroscience, “Dino Ferrari” Center, Università degli Studi di Milano, IRCCS Istituto Auxologico Italiano, Milano, ItalyAbstract: Neurodegenerative diseases are a growing public health challenge, and amyotrophic lateral sclerosis (ALS) remains a fatal incurable disease. The advent of stem cell therapy has opened new horizons for both researchers and ALS patients, desperately looking for a treatment. ALS must be considered a systemic disease affecting many cell phenotypes besides motor neurons, even outside the central nervous system. Cell replacement therapy needs to address the specific neurobiological issues of ALS to safely and efficiently reach clinical settings. Moreover, the enormous potential of induced pluripotent cells directly derived from patients for modeling and understanding the pathological mechanisms, in correlation with the discoveries of new genes and animal models, provides new opportunities that need to be integrated with previously described transplantation strategies. Finally, a careful evaluation of preclinical data in conjunction with wary patient choice in clinical trials needs to be established in order to generate meaningful results.Keywords: amyotrophic lateral sclerosis, regenerative medicine, stem cell therapy, clinical trial

Human Vasculogenesis Ex Vivo: Embryonal Aorta as a Tool for Isolation of Endothelial Cell Progenitors

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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

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

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

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