Sorting live stem cells based on Sox2 mRNA expression.

PMCID: PMC3507951This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs) offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES) and neural stem cells (NSC). One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+)SSEA1(+) cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+) cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(-) cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner

Synthesis of 1,8-naphthalimide-based probes with fluorescent switch triggered by flufenamic acid

This work was supported by the European Commission Marie Curie IEF NANOGEND (ga 299266) to DV and the European Commission Marie Curie ITN NANODRUG (ga 289454) to GS

Fabrication and characterization of bioresorbable, electroactive and highly regular nanomodulated cell interfaces

Biomaterial-based implantable scaffolds capable of promoting physical and functional reconnection of injured spinal cord and nerves represent the latest frontier in neural tissue engineering. Here, we report the fabrication and characterization of self-standing, biocompatible and bioresorbable substrates endowed with both controlled nanotopography and electroactivity, intended for the design of transient implantable scaffolds for neural tissue engineering. In particular, we obtain conductive and nano-modulated poly(D,L-lactic acid) (PLA) and poly(lactic-co-glycolic acid) free-standing films by simply iterating a replica moulding process and coating the polymer with a thin layer of poly(3,4-ethylendioxythiophene) polystyrene sulfonate. The capability of the substrates to retain both surface patterning and electrical properties when exposed to a liquid environment has been evaluated by atomic force microscopy, electrochemical impedance spectroscopy and thermal characterizations. In particular, we show that PLA-based films maintain their surface nano-modulation for up to three weeks of exposure to a liquid environment, a time sufficient for promoting axonal anisotropic sprouting and growth during neuronal cell differentiation. In conclusion, the developed substrates represent a novel and easily-tunable platform to design bioresorbable implantable devices featuring both topographic and electrical cues

Positioning accuracy and facet joints violation after percutaneous pedicle screws placement with robot-assisted versus fluoroscopy-guided technique: Systematic review and meta-analysis

Introduction: Minimally invasive spine surgery became the gold standard for the treatment of many spinal diseases. Only a few comparative studies were performed regarding the superiority of robotic-assisted (RA) surgery over fluoroscopic guidance (FG) surgery during percutaneous pedicle screws placement. Therefore, the aim of the present study was to conduct a systematic literature review and meta-analysis to evaluate the accuracy and potential advantages of RA compared with FG. Material and Methods: This study is a systematic literature review conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. The review questions were formulated following the PICO scheme. Measured outcomes were presented using Forest plots. Heterogeneity among the included studies was assessed using the χ2 test, and the I2 statistic was utilized to estimate the proportion of total variation among the studies. A value exceeding 50% was considered indicative of substantial heterogeneity. Results: Seven studies that met inclusion criteria were finally included in this meta-analysis. These seven studies include: 447 patients, 228 patients (931 screws) treated with robotic guide, and 219 patients (767 pedicle screws) using fluoroscopic guide, with a mean age of 55.2. The percentages of clinically acceptable screws were 94.3% in the robot-assisted group and 89% in the fluoroscopic guided group. The percentages of non-acceptable screws were 5.7% in the robot-assisted group and 11% in the fluoroscopic-guided group. Discussion: Significant differences were observed between the two groups in terms of radiographic and clinical outcomes, with the robotic-assisted pedicle screw group exhibiting longer operative times. Robot technology serves as a valuable tool for assisting surgeons in challenging scenarios such as anatomical variants or patients with spinal deformities, ensuring accurate screw placement. Conclusion: The accuracy of pedicle screw placement with robotic technology is higher than with FG. In fact, the robotic approach allows significantly lower complication rates, fewer cases of violation of the proximal articular facet, less intraoperative exposure to radiation, even if it requires longer surgical times than the FG technique

PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: cyclosporin A as an example

Micelles formed from amphiphilic block copolymers have been explored in recent years as carriers for hydrophobic drugs. In an aqueous environment, the hydrophobic blocks form the core of the micelle, which can host lipophilic drugs, while the hydrophilic blocks form the corona or outer shell and stabilize the interface between the hydrophobic core and the external medium. In the present work, mesophase behavior and drug encapsulation were explored in the AB block copolymeric amphiphile composed of poly(ethylene glycol) (PEG) as a hydrophile and poly(propylene sulfide) PPS as a hydrophobe, using the immunosuppressive drug cyclosporin A (CsA) as an example of a highly hydrophobic drug. Block copolymers with a degree of polymerization of 44 on the PEG and of 10, 20 and 40 on the PPS respectively (abbreviated as PEG44-b-PPS10, PEG44-b-PPS20, PEG44-b-PPS40) were synthesized and characterized. Drug-loaded polymeric micelles were obtained by the cosolvent displacement method as well as the remarkably simple method of dispersing the warm polymer melt, with drug dissolved therein, in warm water. Effective drug solubility up to 2 mg/mL in aqueous media was facilitated by the PEG- b-PPS micelles, with loading levels up to 19% w/w being achieved. Release was burst-free and sustained over periods of 9-12 days. These micelles demonstrate interesting solubilization characteristics, due to the low glass transition temperature, highly hydrophobic nature, and good solvent properties of the PPS bloc

Engineering Mesenchymal Stem Cells with Nanomaterials (MSC-NANO): a novel immunotherapeutic platform to avoid rejection in pancreatic islet transplantations

Islet transplantation is a beta-cell replacement therapy for patients with type 1 diabetes (T1D). Despite the advances, the clinical use of beta-cell transplantation is considerably limited by the need for lifelong immunosuppression to prevent rejection, which is unavoidably accompanied by serious side effects. Our research focuses on developing novel immunotherapies using our biocompatible and biodegradable platform of nanoparticles named drug-integrating amphiphilic nanomaterial assemblies (DIANAs). DIANAs can load high amounts of immunosuppressant drug (e.g. Rapamycin) and be internalized by any kind of cells, including stem cells, without causing toxicity. Our objective was to demonstrate that the combination of DIANAs with immunomodulatory cells (MSC) maximizes immunoprotection of transplanted islets without systemic side effects. DIANAs with shape and size of elongated nano-fibrils (nFIB) were prepared by self-assembling of PEG-OES block copolymer and were incubated with MSC for 24h. Formation of stable MSC-NANO was confirmed by fluorescence analysis. MSC-NANO containing the immunosuppressant Rapamycin (RAPA) were also prepared simply pre-loading the drug in the inner core of the nFIB. MSC-NANO-RAPA were aggregated on human pancreatic islets and cocultured with human activated T cells (PBMCs). To demonstrate that MSC-NANO-RAPA can protect islets from immune attacks, the proliferation inhibition of T cells was evaluated by flow cytometry and the islet functionality by insulin secretion assay. In vivo, the ability of MSC-NANO to prolong the survival of allogeneic transplants was assessed in chemically induced diabetic mice transplanted with Islet/MSC-NANO-RAPA clusters. Mice blood glucose level was daily monitored to determined graft survival in MSC-NANO treated vs. control groups. MSC-NANO stably aggregated with pancreatic islets (Fig.1) and allowed controlled and sustained localized release of RAPA potentiating the MSC ability of inhibiting activated T cells proliferation and promoting Treg expansion. In vivo, co-implanting MSC-NANO-RAPA with allogeneic islet cells in preclinical models of type 1 diabetes showed prolonged survival of the graft (Fig. 2). MSC-NANO demonstrated to be a promising new therapeutic platform to treat T1D. Our goal is having the graft surviving for 150 days post operation and to confirm local immunomodulation by investigating the immune response in draining lymph nodes in order to translate the platform to clinical studies