Erratum: Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

[This corrects the article DOI: 10.1016/j.omtm.

Immortalized human myoblast cell lines for the delivery of therapeutic proteins using encapsulated cell technology.

Despite many promising results obtained in previous preclinical studies, the clinical development of encapsulated cell technology (ECT) for the delivery of therapeutic proteins from macrocapsules is still limited, mainly due to the lack of an allogeneic cell line compatible with therapeutic application in humans. In our work, we generated an immortalized human myoblast cell line specifically tailored for macroencapsulation. In the present report, we characterized the immortalized myoblasts and described the engineering process required for the delivery of functional therapeutic proteins including a cytokine, monoclonal antibodies and a viral antigen.

Endogenous erythropoietin signaling regulates migration and laminar positioning of upper-layer neurons in the developing neocortex.

Erythropoietin (EPO), the hypoxia-inducible hematopoietic hormone, has well-established neuroprotective/neurotrophic roles in the developing central nervous system and the therapeutic potential of EPO has been widely explored in clinical studies for the treatment of perinatal hypoxic brain lesion, as well as prematurity. Here, we reveal that both EPO and Epo receptor (EPOR) are expressed in the developing rat somatosensory cortex during radial migration and laminar positioning of granular and supragranular neurons. Experimental deregulation of EPO signaling using genetic approaches results in aberrant migration, as well as permanent neuronal misplacement leading to abnormal network activity and protracted sensory behavioral deficits.

Transplanted Embryonic Neurons Improve Functional Recovery by Increasing Activity in Injured Cortical Circuits.

Transplantation of appropriate neuronal precursors after injury is a promising strategy to reconstruct cortical circuits, but the efficiency of these approaches remains limited. Here, we applied targeted apoptosis to selectively ablate layer II/III pyramidal neurons in the rat juvenile cerebral cortex and attempted to replace lost neurons with their appropriate embryonic precursors by transplantation. We demonstrate that grafted precursors do not migrate to replace lost neurons but form vascularized clusters establishing reciprocal synaptic contacts with host networks and show functional integration.

Transient Deregulation of Canonical Wnt Signaling in Developing Pyramidal Neurons Leads to Dendritic Defects and Impaired Behavior.

During development, the precise implementation of molecular programs is a key determinant of proper dendritic development. Here, we demonstrate that canonical Wnt signaling is active in dendritic bundle-forming layer II pyramidal neurons of the rat retrosplenial cortex during dendritic branching and spine formation. Transient downregulation of canonical Wnt transcriptional activity during the early postnatal period irreversibly reduces dendritic arbor architecture, leading to long-lasting deficits in spatial exploration and/or navigation and spatial memory in the adult.

Optimizing Synthetic miRNA Minigene Architecture for Efficient miRNA Hairpin Concatenation and Multi-target Gene Knockdown.

Synthetic microRNA (miRNA) minigenes (SMIGs) have a major potential for molecular therapy; however, their optimal architecture still needs to be determined. We have previously optimized the stem structure of miRNA hairpins for efficient gene knockdown. Here, we investigate the overall architecture of SMIGs driven by polymerase II-dependent promoters.

Perturbed Wnt signaling leads to neuronal migration delay, altered interhemispheric connections and impaired social behavior.

Perturbed neuronal migration and circuit development have been implicated in the pathogenesis of neurodevelopmental diseases; however, the direct steps linking these developmental errors to behavior alterations remain unknown. Here we demonstrate that Wnt/C-Kit signaling is a key regulator of glia-guided radial migration in rat somatosensory cortex. Transient downregulation of Wnt signaling in migrating, callosal projection neurons results in delayed positioning in layer 2/3.

EMMPRIN overexpression in SVZ neural progenitor cells increases their migration towards ischemic cortex.

Stimulation of endogenous neurogenesis and recruitment of neural progenitors from the subventricular zone (SVZ) neurogenic site may represent a useful strategy to improve regeneration in the ischemic cortex. Here, we tested whether transgenic overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN), the regulator of matrix metalloproteinases (MMPs) expression, in endogenous neural progenitor cells (NPCs) in the subventricular zone (SVZ) could increase migration towards ischemic injury. For this purpose, we applied a lentivector-mediated gene transfer system.

Multimodal MRI Imaging of Apoptosis-Triggered Microstructural Alterations in the Postnatal Cerebral Cortex.

Prematurely born children often develop neurodevelopmental delay that has been correlated with reduced growth and microstructural alterations in the cerebral cortex. Much research has focused on apoptotic neuronal cell death as a key neuropathological features following preterm brain injuries. How scattered apoptotic death of neurons may contribute to microstructural alterations remains unknown.

Elimination of proliferating cells from CNS grafts using a Ki67 promoter-driven thymidine kinase.

Pluripotent stem cell (PSC)-based cell therapy is an attractive concept for neurodegenerative diseases, but can lead to tumor formation. This is particularly relevant as proliferating neural precursors rather than postmitotic mature neurons need to be transplanted. Thus, safety mechanisms to eliminate proliferating cells are needed.

Astrocytes spatially restrict VEGF signaling by polarized secretion and incorporation of VEGF into the actively assembling extracellular matrix.

The spatial organization of vascular endothelial growth factor (VEGF) signaling is a key determinant of vascular patterning during development and tissue repair. How VEGF signaling becomes spatially restricted and the role of VEGF secreting astrocytes in this process remains poorly understood. Using a VEGF-GFP fusion protein and confocal time-lapse microscopy, we observed the intracellular routing, secretion and immobilization of VEGF in scratch-activated living astrocytes.

Apoptotic neurons induce proliferative responses of progenitor cells in the postnatal neocortex.

Apoptotic cell death is the leading cause of neuronal loss after neonatal brain injury. Little is known about the intrinsic capacity of the immature cerebral cortex for replacing dead cells. Here we test the hypothesis that neuronal apoptosis is able to trigger compensatory proliferation in surrounding cells.

Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice.

Unlabelled: Gene-engineered CD34(+) hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34(+) cells.

Wnt signaling regulates multipolar-to-bipolar transition of migrating neurons in the cerebral cortex.

The precise timing of pyramidal cell migration from the ventricular germinal zone to the cortical plate is essential for establishing cortical layers, and migration errors can lead to neurodevelopmental disorders underlying psychiatric and neurological diseases. Here, we report that Wnt canonical as well as non-canonical signaling is active in pyramidal precursors during radial migration. We demonstrate using constitutive and conditional genetic strategies that transient downregulation of canonical Wnt/β-catenin signaling during the multipolar stage plays a critical role in polarizing and orienting cells for radial migration.

Optimization of Critical Hairpin Features Allows miRNA-based Gene Knockdown Upon Single-copy Transduction.

Gene knockdown using micro RNA (miRNA)-based vector constructs is likely to become a prominent gene therapy approach. It was the aim of this study to improve the efficiency of gene knockdown through optimizing the structure of miRNA mimics. Knockdown of two target genes was analyzed: CCR5 and green fluorescent protein.

Overexpression of E2F3 promotes proliferation of functional human β cells without induction of apoptosis.

The mechanisms that control proliferation, or lack thereof, in adult human β cells are poorly understood. Controlled induction of proliferation could dramatically expand the clinical application of islet cell transplantation and represents an important component of regenerative approaches to a functional cure of diabetes. Adult human β cells are particularly resistant to common proliferative targets and often dedifferentiate during proliferation.

The European Medicines Agency approval of 5-aminolaevulinic acid (Ameluz) for the treatment of actinic keratosis of mild to moderate intensity on the face and scalp: summary of the scientific assessment of the Committee for Medicinal Products for Human Use.

The European Commission has recently issued a marketing authorisation valid throughout the European Union for 5-aminolaevulinic acid (Ameluz). The decision was based on the favorable opinion of the CHMP recommending a marketing authorization for 5-aminolaevulinic acid for treatment of actinic keratosis of mild to moderate intensity on the face and scalp. The active substance is a sensitizer used in photodynamic/radiation therapy (ATC code L01XD04).

Generation of human cell lines using lentiviral-mediated genetic engineering.

Even now, most human cell lines used in research are derived from tumor cells. They are still widely used because they grow well in vitro and so far have helped answering several basic biological questions. However, as modern biology moves into more sophisticated areas, scientists now need human cell lines closer to normal primary cells and further from transformed cancerous cells.

Polyswitch lentivectors: "all-in-one" lentiviral vectors for drug-inducible gene expression, live selection, and recombination cloning.

Lentiviral vectors are now widely considered one of the safest and most efficient tools for gene delivery and stable gene expression. Even though inducible gene expression cassettes are mandatory for many genetic engineering strategies, most current systems suffer from various issues, such as the requirement of two vectors, which decreases the overall efficiency of the transduction, leakiness and/or insufficient levels of activation of the inducible promoter, lack of selectable marker, low titers, or general issues associated with the cloning of large plasmids. In this article, we describe the design and functional characterization of a set of "all-in-one" multicistronic autoinducible lentivectors.

Early postnatal migration and development of layer II pyramidal neurons in the rodent cingulate/retrosplenial cortex.

The cingulate and retrosplenial regions are major components of the dorsomedial (dm) limbic cortex and have been implicated in a range of cognitive functions such as emotion, attention, and spatial memory. While the structure and connectivity of these cortices are well characterized, little is known about their development. Notably, the timing and mode of migration that govern the appropriate positioning of late-born neurons remain unknown.

Lentiviral vectors.

Lentiviral vectors have evolved over the last decade as powerful, reliable, and safe tools for stable gene transfer in a wide variety of mammalian cells. Contrary to other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells. In particular, lentivectors (LVs) derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility.

European regulation on orphan medicinal products: 10 years of experience and future perspectives.

In 2000, regulation on orphan medicinal products was adopted in the European Union with the aim of benefiting patients who suffer from serious, rare conditions for which there is currently no satisfactory treatment. Since then, more than 850 orphan drug designations have been granted by the European Commission based on a positive opinion from the Committee for Orphan Medicinal Products (COMP), and more than 60 orphan drugs have received marketing authorization in Europe. Here, stimulated by the tenth anniversary of the COMP, we reflect on the outcomes and experience gained in the past decade, and contemplate issues for the future, such as catalysing drug development for the large number of rare diseases that still lack effective treatments.

Production and titration of lentiviral vectors.

Lentiviral vectors have emerged over the last decade as powerful, reliable, and safe tools for stable gene transfer in a wide variety of mammalian cells. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells, including neurons. This is why lentivectors (LVs) are becoming the most useful and promising tools in the field of neuroscience, not only for research, but also for future gene and cell therapy approaches.

A software solution for recording circadian oscillator features in time-lapse live cell microscopy.

Background: Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity.

Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells.

Background: Feeder cells are frequently used for the early-stage of derivation and culture of human embryonic stem cell (hESC) lines.

Methods: We established a conditionally immortalized human foreskin fibroblast line that secreted basic fibroblast growth factor (bFGF). These cells were used as feeder cells for hESC culture and induced pluripotent stem (iPS) cell derivation and expansion.