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

A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

We present a full-length α(1)β(2)γ(2) GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and the backbone of β(2)S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α(1)R66, β(2)T202, α(1)T129, β(2)E155, β(2)Y205 and β(2)F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β(2)S156 and β(2)Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α(1)T206 and γ(2)T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α(1)H101 and the N-methyl group near α(1)Y159, α(1)T206, and α(1)Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABA(A) receptor is made available as Model S1

Hydrogel-based scaffolds to support intrathecal stem cell transplantation as a gateway to the spinal cord: clinical needs, biomaterials, and imaging technologies

The prospects for cell replacement in spinal cord diseases are impeded by inefficient stem cell delivery. The deep location of the spinal cord and complex surgical access, as well as densely packed vital structures, question the feasibility of the widespread use of multiple spinal cord punctures to inject stem cells. Disorders characterized by disseminated pathology are particularly appealing for the distribution of cells globally throughout the spinal cord in a minimally invasive fashion. The intrathecal space, with access to a relatively large surface area along the spinal cord, is an attractive route for global stem cell delivery, and, indeed, is highly promising, but the success of this approach relies on the ability of cells 1) to survive in the cerebrospinal fluid (CSF), 2) to adhere to the spinal cord surface, and 3) to migrate, ultimately, into the parenchyma. Intrathecal infusion of cell suspension, however, has been insufficient and we postulate that embedding transplanted cells within hydrogel scaffolds will facilitate reaching these goals. In this review, we focus on practical considerations that render the intrathecal approach clinically viable, and then discuss the characteristics of various biomaterials that are suitable to serve as scaffolds. We also propose strategies to modulate the local microenvironment with nanoparticle carriers to improve the functionality of cellular grafts. Finally, we provide an overview of imaging modalities for in vivo monitoring and characterization of biomaterials and stem cells. This comprehensive review should serve as a guide for those planning pre-clinical and clinical studies on intrathecal stem cell transplantation.Funds provided under the project NanoTech4ALS (ref. ENMed/0008/2015, 13/EuroNanoMed/2016), funded under the EU FP7 M-ERA.NET program, Strategmed 1/233209/12/NCBIR/2015, and NIH R01 NS091100. The FCT distinction attributed to J.M.O. under the Investigator FCT program (IF/01285/2015) is also gratefully acknowledgedinfo:eu-repo/semantics/publishedVersio

Reparative effects of neural stem cells in neonatal rats with hypoxic–ischemic injury are not influenced by host sex

BackgroundGender is increasingly recognized as an important influence on brain development, disease susceptibility, and response to pharmacologic/rehabilitative treatments. In regenerative medicine, it remains entirely unknown whether there is an interaction between transplanted stem cells and host gender that might bias efficacy and safety in some patients but not others.MethodsWe examined the role of recipient gender in a neonatal rat hypoxic-ischemic injury (HII) model, treated with female human neuronal stem cells (hNSCs), labeled with superparamagnetic iron oxide particles implanted into the contralateral cerebral ventricle. We monitored HII evolution (by magnetic resonance imaging, histopathology, behavioral testing) and hNSC fate (migration, replication, viability).ResultsRecipient gender after implantation did not influence the volume or location of ischemic injury (1, 30, or 90 d) or behavior (90 d). Superparamagnetic iron oxide labeling did not influence HII evolution. Implantation had its greatest benefit on mild/moderate injuries, which remained stable rather than increasing as in severe HII as is the natural history for such lesions.ConclusionOur results suggest that hNSC treatment (including using hNSCs that are prelabeled with iron to allow tracking in real time by magnetic resonance imaging) would be equally safe and effective for male and female human newborns with mild-to-moderate HII

Optimization of the magnetic labeling of human neural stem cells and MRI visualization in the hemiparkinsonian rat brain

[Background] Magnetic resonance imaging is the ideal modality for non-invasive in vivo cell tracking allowing for longitudinal studies over time. Cells labeled with superparamagnetic iron oxide nanoparticles have been shown to induce sufficient contrast for in vivo magnetic resonance imaging enabling the in vivo analysis of the final location of the transplanted cells. For magnetic nanoparticles to be useful, a high internalization efficiency of the particles is required without compromising cell function, as well as validation of the magnetic nanoparticles behaviour inside the cells.[Results] In this work, we report the development, optimization and validation of an efficient procedure to label human neural stem cells with commercial nanoparticles in the absence of transfection agents. Magnetic nanoparticles used here do not affect cell viability, cell morphology, cell differentiation or cell cycle dynamics. Moreover, human neural stem cells progeny labeled with magnetic nanoparticles are easily and non-invasively detected long time after transplantation in a rat model of Parkinson's disease (up to 5 months post-grafting) by magnetic resonance imaging.[Conclusions+ These findings support the use of commercial MNPs to track cells for short- and mid-term periods after transplantation for studies of brain cell replacement therapy. Nevertheless, long-term MR images should be interpreted with caution due to the possibility that some MNPs may be expelled from the transplanted cells and internalized by host microglial cells.This work was supported by grants from (to AM-S): Spanish Ministry of Economy and Competitiveness (SAF2010-17167), Comunidad Autónoma Madrid (S2011-BMD-2336), Instituto Salud Carlos III (RETICS TerCel, RD12/0019/ 0013). This work was also supported by an institutional grant from Fundación Ramón Areces to the Center of Molecular Biology Severo Ochoa. The authors gratefully acknowledge the financial support of the Reina Sofia Foundation and Comunidad Autónoma Madrid (S2010-BMD-2460) to MR-G.Peer Reviewe