Encapsulation of MSCs and GDNF in an Injectable Nanoreinforced Supramolecular Hydrogel for Brain Tissue Engineering

The co-administration of glial cell line-derived neurotrophic factor (GDNF) and mesenchymal stem cells (MSCs) in hydrogels (HGs) has emerged as a powerful strategy to enhance the efficient integration of transplanted cells in Parkinson's disease (PD). This strategy could be improved by controlling the cellular microenvironment and biomolecule release and better mimicking the complex properties of the brain tissue. Here, we develop and characterize a drug delivery system for brain repair where MSCs and GDNF are included in a nanoparticle-modified supramolecular guest-host HA HG. In this system, the nanoparticles act as both carriers for the GDNF and active physical crosslinkers of the HG. The multifunctional HG is mechanically compatible with brain tissue and easily injectable. It also protects GDNF from degradation and achieves its controlled release over time. The cytocompatibility studies show that the developed biomaterial provides a friendly environment for MSCs and presents good compatibility with PC12 cells. Finally, using RNA-sequencing (RNA-seq), we investigated how the three-dimensional (3D) environment, provided by the nanostructured HG, impacted the encapsulated cells. The transcriptome analysis supports the beneficial effect of including MSCs in the nanoreinforced HG. An enhancement in the anti-inflammatory effect of MSCs was observed, as well as a differentiation of the MSCs toward a neuron-like cell type. In summary, the suitable strength, excellent self healing properties, good biocompatibility, and ability to boost MSC regenerative potential make this nanoreinforced HG a good candidate for drug and cell administration to the brain

Development of three-dimensional biomaterials containing stem cells and neurotrophic factors for brain repair in Parkinson's disease

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Development of three-dimensional biomaterials containing stem cells and neurotrophic factors for brain repair in Parkinson's disease

Therapeutical management of Parkinson's disease (PD) is predominantly focused on controlling motor symptoms and there is no gold standard treatment strategy. Consequently, the medical treatment is tailored to each patient, based on the severity of their symptoms and the adverse effects associated with their medication. Unfortunately, the available therapies provide temporary relief of symptoms but are unable to reverse and halt the disease progression. In the light of this situation, this thesis addresses the development of a PD-modifying therapy where stem cells and the glial cell line-derived neurotrophic factor (hGDNF) are included in a nanoparticle-modified HG. To this end, the following challenges were investigated: 1)The production of clinical-grade therapeutic hGDNF to provide appropriate post-translational modifications and to solve the safety issues related to the immune response (Chapter 1). 2)The assessment of hGDNF potential to modulate the gene expression profile of two cell types with potential for clinical use in PD: Dopaminergic neurons derived from human induced pluripotent stem cells (hiPS-DAns) and human mesenchymal stem cells (hMSCs). (Chapter 2) 3)The development and characterization of an original drug delivery system for brain tissue engineering, where hGDNF and hMSCs are rationally combined into a nanoreinforced HG for their simultaneous administration into the brain (Chapter 2). In the introduction, some of the most encouraging micro- and nanotechnology advances for PD application are summarized and discussed. The experimental section of this thesis is divided into two chapters. The first experimental section presents a novel biphasic temperature cultivation protocol to improve the expression of hGDNF with a high degree of purity and a specific glycosylation pattern. In these experiments, an RNA vector was electroporated into the baby hamster kidney cell line and the electroporated cells were incubated at 37ºC or 33ºC with 5% CO2. A significant improvement in cell survival and hGDNF expression was demonstrated with the increase in the temperature from 33ºC to 37 ºC during the shut-off period . In consonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Subsequently, in the second chapter, the potential of hGDNF to induce changes in the transcriptome of two cell types with potential for clinical use in PD (hiPS-DAns and hMSCs) was evaluated. The observed effects suggest that this neurotrophic factor can stimulate the expression of genes involved in neuronal plasticity and neurogenesis in both cell types. Next, the neurotrophic factor was successfully encapsulated in polymeric NPs to be subsequently included in a HA-HG modified with adamantane and cyclodextrin. In this section, the mechanical properties of this scaffold were investigated. Then, the neural compatibility of the system was studied on PC12 cells and its compatibility with the hMSCs cell line was explored. Regarding mechanical properties, the developed biomaterial demonstrated shear-thinning and self-healing features. Moreover, the NPs incorporation into the HG allowed a more sustained hGDNF release profile, as well as a significant reduction of drug release at two weeks. Importantly, this scaffold provided an ideal environment for PC12 and hMSCs. In summary, the suitable strength, excellent self-healing properties and good biocompatibility make this HG a good candidate to administer drugs and cells for brain repair applications. Finally, a comprehensive perspective on the different hypotheses set forth in this thesis as well as their scientific contribution to the brain regenerative field is provided in the General Discussion

Optimization of a GDNF production method based on Semliki Forest virus vector

Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson’s disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33◦C to 37◦C during the “shut-off period”, produced a significant improvement in cell survival and hGDNF expression. Inconsonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a “recovery period” at 37◦C before cells are exposed at 33◦C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF

Optimization of a GDNF production method based on Semliki Forest virus vector

Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson’s disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33◦C to 37◦C during the “shut-off period”, produced a significant improvement in cell survival and hGDNF expression. Inconsonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a “recovery period” at 37◦C before cells are exposed at 33◦C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF