Optimization of Thymidine Kinase-Based Safety Switch for Neural Cell Therapy.

Cell therapies based on pluripotent stem cells (PSC), have opened new therapeutic strategies for neurodegenerative diseases. However, insufficiently differentiated PSC can lead to tumor formation. Ideally, safety switch therapies should selectively kill proliferative transplant cells while preserving post-mitotic neurons. In this study, we evaluated the potential of nucleoside analogs and thymidine kinase-based suicide genes. Among tested thymidine kinase variants, the humanized SR39 (SR39h) variant rendered cells most sensitive to suicide induction. Unexpectedly, post-mitotic neurons with ubiquitous SR39h expression were killed by ganciclovir, but were spared when SR39h was expressed under the control of the cell cycle-dependent Ki67 promoter. The efficacy of six different nucleoside analogs to induce cell death was then evaluated. Penciclovir (PCV) showed the most interesting properties with an efficiency comparable to ganciclovir (GCV), but low toxicity. We tested three nucleoside analogs in vivo: at concentrations of 40 mg/kg/day, PCV and GCV prevented tumor formation, while acyclovir (ACV) did not. In summary, SR39h under the control of a cell cycle-dependent promoter appears most efficient and selective as safety switch for neural transplants. In this setting, PCV and GCV are efficient inducers of cell death. Because of its low toxicity, PCV might become a preferred alternative to GCV.

Neurothreads: Development of supportive carriers for mature dopaminergic neuron differentiation and implantation.

In this study we present the use of elastic macroporous cryogels for differentiation and transplantation of mature neurons. We develop a coating suitable for long-term neuronal culture, including stem cell differentiation, by covalent immobilization of neural adhesion proteins. In the context of cell therapy for Parkinson's disease, we show compatibility with established dopaminergic differentiation of both immortalized mesencephalic progenitors - LUHMES - and human embryonic stem cells (hESCs). We adjust structural properties of the biomaterial to create carriers - Neurothreads - favourable for cell viability during transplantation. Finally, we show feasibility of preservation of mature neurons, supported by Neurothreads, one month after in-vivo transplantation. Preliminary data suggests that the Neurothread approach could provide more mature and less proliferative cells in vivo.

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

Host Cell Copper Transporters CTR1 and ATP7A are important for Influenza A virus replication.

BACKGROUND: The essential role of copper in eukaryotic cellular physiology is known, but has not been recognized as important in the context of influenza A virus infection. In this study, we investigated the effect of cellular copper on influenza A virus replication. METHODS: Influenza A/WSN/33 (H1N1) virus growth and macromolecule syntheses were assessed in cultured human lung cells (A549) where the copper concentration of the growth medium was modified, or expression of host genes involved in copper homeostasis was targeted by RNA interference. RESULTS: Exogenously increasing copper concentration, or chelating copper, resulted in moderate defects in viral growth. Nucleoprotein (NP) localization, neuraminidase activity assays and transmission electron microscopy did not reveal significant defects in virion assembly, morphology or release under these conditions. However, RNAi knockdown of the high-affinity copper importer CTR1 resulted in significant viral growth defects (7.3-fold reduced titer at 24 hours post-infection, p = 0.04). Knockdown of CTR1 or the trans-Golgi copper transporter ATP7A significantly reduced polymerase activity in a minigenome assay. Both copper transporters were required for authentic viral RNA synthesis and NP and matrix (M1) protein accumulation in the infected cell. CONCLUSIONS: These results demonstrate that intracellular copper regulates the influenza virus life cycle, with potentially distinct mechanisms in specific cellular compartments. These observations provide a new avenue for drug development and studies of influenza virus pathogenesis.