Miniaturization of hiPSC-derived 3D neural cultures in stirred-tank bioreactors for parallelized preclinical assessment of rAAV.

Introduction: Engineered 3D models employing human induced pluripotent stem cell (hiPSC) derivatives have the potential to recapitulate the cell diversity and structure found in the human central nervous system (CNS). Therefore, these complex cellular systems offer promising human models to address the safety and potency of advanced therapy medicinal products (ATMPs), such as gene therapies. Specifically, recombinant adeno-associated viruses (rAAVs) are currently considered highly attractive for CNS gene therapy due to their broad tropism, low toxicity, and moderate immunogenicity. To accelerate the clinical translation of rAAVs, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. The integration of hiPSC-derived CNS models in rAAV development will require, amongst other factors, robust, small-scale, high-throughput culture platforms that can feed the preclinical trials. Methods: Herein, we pioneer the miniaturization and parallelization of a 200 mL stirred-tank bioreactor-based 3D brain cell culture derived from hiPSCs. We demonstrate the applicability of the automated miniaturized Ambr® 15 Cell Culture system for the maintenance of hiPSC-derived neurospheroids (iNSpheroids), composed of neuronal and glial cells. Critical process parameters were optimized, namely, cell density and agitation mode. Results: Under optimized conditions, stable iNSpheroid cultures were attained in the microbioreactors for at least 15 days, with high cell viability and astrocytic and neuronal phenotype maintenance. This culture setup allowed the parallelization of different rAAVs, in different multiplicity of infections (MOIs), to address rAAV-host interactions at a preclinical scale. The iNSpheroids were exposed to rAAV2- and rAAV9-eGFP in the microbioreactors. Transgene expression was detected 14 days post-transduction, revealing different astrocyte/neuron tropism of the two serotypes. Discussion: We advocate that the iNSpheroid cultures in miniaturized bioreactors are reliable and reproducible screening tools for addressing rAAV transduction and tropism, compatible with preclinical demands.

Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay.

Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.

Novel scFv against Notch Ligand JAG1 Suitable for Development of Cell Therapies toward JAG1-Positive Tumors.

The Notch signaling ligand JAG1 is overexpressed in various aggressive tumors and is associated with poor clinical prognosis. Hence, therapies targeting oncogenic JAG1 hold great potential for the treatment of certain tumors. Here, we report the identification of specific anti-JAG1 single-chain variable fragments (scFvs), one of them endowing chimeric antigen receptor (CAR) T cells with cytotoxicity against JAG1-positive cells. Anti-JAG1 scFvs were identified from human phage display libraries, reformatted into full-length monoclonal antibodies (Abs), and produced in mammalian cells. The characterization of these Abs identified two specific anti-JAG1 Abs (J1.B5 and J1.F1) with nanomolar affinities. Cloning the respective scFv sequences in our second- and third-generation CAR backbones resulted in six anti-JAG1 CAR constructs, which were screened for JAG1-mediated T-cell activation in Jurkat T cells in coculture assays with JAG1-positive cell lines. Studies in primary T cells demonstrated that one CAR harboring the J1.B5 scFv significantly induced effective T-cell activation in the presence of JAG1-positive, but not in JAG1-knockout, cancer cells, and enabled specific killing of JAG1-positive cells. Thus, this new anti-JAG1 scFv represents a promising candidate for the development of cell therapies against JAG1-positive tumors.

Transcriptome and proteome profiling of activated cardiac fibroblasts supports target prioritization in cardiac fibrosis.

Background: Activated cardiac fibroblasts (CF) play a central role in cardiac fibrosis, a condition associated with most cardiovascular diseases. Conversion of quiescent into activated CF sustains heart integrity upon injury. However, permanence of CF in active state inflicts deleterious heart function effects. Mechanisms underlying this cell state conversion are still not fully disclosed, contributing to a limited target space and lack of effective anti-fibrotic therapies. Materials and methods: To prioritize targets for drug development, we studied CF remodeling upon activation at transcriptomic and proteomic levels, using three different cell sources: primary adult CF (aHCF), primary fetal CF (fHCF), and induced pluripotent stem cells derived CF (hiPSC-CF). Results: All cell sources showed a convergent response upon activation, with clear morphological and molecular remodeling associated with cell-cell and cell-matrix interactions. Quantitative proteomic analysis identified known cardiac fibrosis markers, such as FN1, CCN2, and Serpine1, but also revealed targets not previously associated with this condition, including MRC2, IGFBP7, and NT5DC2. Conclusion: Exploring such targets to modulate CF phenotype represents a valuable opportunity for development of anti-fibrotic therapies. Also, we demonstrate that hiPSC-CF is a suitable cell source for preclinical research, displaying significantly lower basal activation level relative to primary cells, while being able to elicit a convergent response upon stimuli.

Capturing the third dimension in drug discovery: Spatially-resolved tools for interrogation of complex 3D cell models.

Advanced three-dimensional (3D) cell models have proven to be capable of depicting architectural and microenvironmental features of several tissues. By providing data of higher physiological and pathophysiological relevance, 3D cell models have been contributing to a better understanding of human development, pathology onset and progression mechanisms, as well as for 3D cell-based assays for drug discovery. Nonetheless, the characterization and interrogation of these tissue-like structures pose major challenges on the conventional analytical methods, pushing the development of spatially-resolved technologies. Herein, we review recent advances and pioneering technologies suitable for the interrogation of multicellular 3D models, while capable of retaining biological spatial information. We focused on imaging technologies and omics tools, namely transcriptomics, proteomics and metabolomics. The advantages and shortcomings of these novel methodologies are discussed, alongside the opportunities to intertwine data from the different tools.

Unveiling dynamic metabolic signatures in human induced pluripotent and neural stem cells.

Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators.

Flexible 3D Cell-Based Platforms for the Discovery and Profiling of Novel Drugs Targeting Plasmodium Hepatic Infection.

The restricted pipeline of drugs targeting the liver stage of Plasmodium infection reflects the scarcity of cell models that mimic the human hepatic phenotype and drug metabolism, as well as Plasmodium hepatic infection. Using stirred-tank culture systems, spheroids of human hepatic cell lines were generated, sustaining a stable hepatic phenotype over 4 weeks of culture. Spheroids were employed in the establishment of 3D Plasmodium berghei infection platforms that relied on static or dynamic culture conditions. P. berghei invasion and development were recapitulated in the hepatic spheroids, yielding blood-infective merozoites. The translational potential of the 3D platforms was demonstrated by comparing the in vitro minimum inhibitory concentration of M5717, a compound under clinical development, with in vivo plasma concentrations that clear liver stage P. berghei in mice. Our results show that the 3D platforms are flexible and scalable and can predict the efficacy of antiplasmodial therapies, constituting a powerful tool for integration in drug discovery programs.

Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neurons.

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient ß-glucuronidase (ß-gluc) activity. Significantly reduced ß-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for ß-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced ß-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant ß-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced ß-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects.

Full-length human CCBE1 production and purification: leveraging bioprocess development for high quality glycosylation attributes and functionality.

Collagen and calcium-binding EGF domain-1 (CCBE1) is a secreted protein critical for lymphatic/cardiac vascular development and regeneration. However, the low efficient production of the recombinant full-length CCBE1 (rCCBE1) has been a setback for functional studies and therapeutic applications using this protein. The main goal of this work was to implement a robust bioprocess for efficient production of glycosylated rCCBE1. Different bioprocess strategies were combined with proteomic tools for process/product characterization, evaluating the impact of process parameters on cell performance, rCCBE1 production and quality. We have shown that rCCBE1 volumetric yield was positively correlated with higher cell density at transfection (HDT), and under these conditions the secreted protein presented a mature glycosylated profile (complex N-glycans). Mild hypothermia was also applied to HDT condition that resulted in enhanced cell viability; however an enrichment of immature rCCBE1 variants was detected. Mass spectrometry-based tools allowed the identification of rCCBE1 peptides confirming protein identity in the affinity chromatography enriched product. rCCBE1 biological activity was validated by in vitro angiogenesis assay, where enhanced vessel formation was observed. Herein, we report a step forward in the production and characterization of human glycosylated rCCBE1, amenable for in vitro and in vivo studies to explore its regenerative therapeutic potential.

Recapitulation of Human Neural Microenvironment Signatures in iPSC-Derived NPC 3D Differentiation.

Brain microenvironment plays an important role in neurodevelopment and pathology, where the extracellular matrix (ECM) and soluble factors modulate multiple cellular processes. Neural cell culture typically relies on heterologous matrices poorly resembling brain ECM. Here, we employed neurospheroids to address microenvironment remodeling during neural differentiation of human stem cells, without the confounding effects of exogenous matrices. Proteome and transcriptome dynamics revealed significant changes at cell membrane and ECM during 3D differentiation, diverging significantly from the 2D differentiation. Structural proteoglycans typical of brain ECM were enriched during 3D differentiation, in contrast to basement membrane constituents in 2D. Moreover, higher expression of synaptic and ion transport machinery was observed in 3D cultures, suggesting higher neuronal maturation in neurospheroids. This work demonstrates that 3D neural differentiation as neurospheroids promotes the expression of cellular and extracellular features found in neural tissue, highlighting its value to address molecular defects in cell-ECM interactions associated with neurological disorders.

Energy-Efficient Channel Coding Strategy for Underwater Acoustic Networks.

Underwater acoustic networks (UAN) allow for efficiently exploiting and monitoring the sub-aquatic environment. These networks are characterized by long propagation delays, error-prone channels and half-duplex communication. In this paper, we address the problem of energy-efficient communication through the use of optimized channel coding parameters. We consider a two-layer encoding scheme employing forward error correction (FEC) codes and fountain codes (FC) for UAN scenarios without feedback channels. We model and evaluate the energy consumption of different channel coding schemes for a K-distributed multipath channel. The parameters of the FEC encoding layer are optimized by selecting the optimal error correction capability and the code block size. The results show the best parameter choice as a function of the link distance and received signal-to-noise ratio.

Functional metabolic interactions of human neuron-astrocyte 3D in vitro networks.

The generation of human neural tissue-like 3D structures holds great promise for disease modeling, drug discovery and regenerative medicine strategies. Promoting the establishment of complex cell-cell interactions, 3D culture systems enable the development of human cell-based models with increased physiological relevance, over monolayer cultures. Here, we demonstrate the establishment of neuronal and astrocytic metabolic signatures and shuttles in a human 3D neural cell model, namely the glutamine-glutamate-GABA shuttle. This was indicated by labeling of neuronal GABA following incubation with the glia-specific substrate [2-(13)C]acetate, which decreased by methionine sulfoximine-induced inhibition of the glial enzyme glutamine synthetase. Cell metabolic specialization was further demonstrated by higher pyruvate carboxylase-derived labeling in glutamine than in glutamate, indicating its activity in astrocytes and not in neurons. Exposure to the neurotoxin acrylamide resulted in intracellular accumulation of glutamate and decreased GABA synthesis. These results suggest an acrylamide-induced impairment of neuronal synaptic vesicle trafficking and imbalanced glutamine-glutamate-GABA cycle, due to loss of cell-cell contacts at synaptic sites. This work demonstrates, for the first time to our knowledge, that neural differentiation of human cells in a 3D setting recapitulates neuronal-astrocytic metabolic interactions, highlighting the relevance of these models for toxicology and better understanding the crosstalk between human neural cells.

Perfusion Stirred-Tank Bioreactors for 3D Differentiation of Human Neural Stem Cells.

Therapeutic breakthroughs in neurological disorders have been hampered by the lack of accurate central nervous system (CNS) models. The development of these models allows the study of the disease onset/progression mechanisms and the preclinical evaluation of new therapeutics. This has traditionally relied on genetically engineered animal models that often diverge considerably from the human phenotype (developmental, anatomic, and physiological) and 2D in vitro cell models, which fail to recapitulate the characteristics of the target tissue (cell-cell and cell-matrix interactions, cell polarity, etc.). Recapitulation of CNS phenotypic and functional features in vitro requires the implementation of advanced culture strategies, such as 3D culture systems, which enable to mimic the in vivo structural and molecular complexity. Models based on differentiation of human neural stem cells (hNSC) in 3D cultures have great potential as complementary tools in preclinical research, bridging the gap between human clinical studies and animal models. The development of robust and scalable processes for the 3D differentiation of hNSC can improve the accuracy of early stage development in preclinical research. In this context, the use of software-controlled stirred-tank bioreactors (STB) provides an efficient technological platform for hNSC aggregation and differentiation. This system enables to monitor and control important physicochemical parameters for hNSC culture, such as dissolved oxygen. Importantly, the adoption of a perfusion operation mode allows a stable flow of nutrients and differentiation/neurotrophic factors, while clearing the toxic by-products. This contributes to a setting closer to the physiological, by mimicking the in vivo microenvironment. In this chapter, we address the technical requirements and procedures for the implementation of 3D differentiation strategies of hNSC, by operating STB under perfusion mode for long-term cultures. This strategy is suitable for the generation of human 3D neural in vitro models, which can be used to feed high-throughput screening platforms, contributing to expand the available in vitro tools for drug screening and toxicological studies.

Transcriptional Response of Human Neurospheres to Helper-Dependent CAV-2 Vectors Involves the Modulation of DNA Damage Response, Microtubule and Centromere Gene Groups.

Brain gene transfer using viral vectors will likely become a therapeutic option for several disorders. Helper-dependent (HD) canine adenovirus type 2 vectors (CAV-2) are well suited for this goal. These vectors are poorly immunogenic, efficiently transduce neurons, are retrogradely transported to afferent structures in the brain and lead to long-term transgene expression. CAV-2 vectors are being exploited to unravel behavior, cognition, neural networks, axonal transport and therapy for orphan diseases. With the goal of better understanding and characterizing HD-CAV-2 for brain therapy, we analyzed the transcriptomic modulation induced by HD-CAV-2 in human differentiated neurospheres derived from midbrain progenitors. This 3D model system mimics several aspects of the dynamic nature of human brain. We found that differentiated neurospheres are readily transduced by HD-CAV-2 and that transduction generates two main transcriptional responses: a DNA damage response and alteration of centromeric and microtubule probes. Future investigations on the biochemistry of processes highlighted by probe modulations will help defining the implication of HD-CAV-2 and CAR receptor binding in enchaining these functional pathways. We suggest here that the modulation of DNA damage genes is related to viral DNA, while the alteration of centromeric and microtubule probes is possibly enchained by the interaction of the HD-CAV-2 fibre with CAR.

Human amniocyte-derived cells are a promising cell host for adenoviral vector production under serum-free conditions.

Recombinant adenovirus vectors (AdVs) have been used for the development of vaccines, as gene therapy vectors and for protein production. Currently, the production of clinical grade batches of recombinant E1-deleted adenovirus type 5 vectors is performed using human-derived HEK293 or PER.C6(®) cell lines. In this work we describe the generation of a new human amniocyte-derived cell line named 1G3 and show that it can be used as a very promising cell host for AdV production in serum-free conditions, allowing for production in high cell density cultures and avoiding the typical cell density effect observed for HEK293. By design, this cell line makes the generation of replication-competent adenovirus during production of E1-deleted AdVs very unlikely. The impact of the culture system (static versus agitated) and AdV infection parameters such as multiplicity of infection, time of harvesting and cell concentration at infection were evaluated and compared with HEK293. Using stirred tanks bioreactors, it was possible to grow 1G3 cells to cell densities of up to 9 × 10(6) cells/mL using serum-free media. Moreover, without a medium exchange step at infection, a three-fold increase in AdV volumetric titers was obtained, as no cell density effect was observed at CCI 3. Overall, our results clearly demonstrate the potential of the human amniocyte-derived newly established cell line 1G3 for AdV production in a serum-free scalable process, paving the way for further process improvements based on fed-batch or perfusion strategies.

Modeling human neural functionality in vitro: three-dimensional culture for dopaminergic differentiation.

Advances in mechanistic knowledge of human neurological disorders have been hindered by the lack of adequate human in vitro models. Three-dimensional (3D) cellular models displaying higher biological relevance are gaining momentum; however, their lack of robustness and scarcity of analytical tools adapted to three dimensions hampers their widespread implementation. Herein we show that human midbrain-derived neural progenitor cells, cultured as 3D neurospheres in stirred culture systems, reproducibly differentiate into complex tissue-like structures containing functional dopaminergic neurons, as well as astrocytes and oligodendrocytes. Moreover, an extensive toolbox of analytical methodologies has been adapted to 3D neural cell models, allowing molecular and phenotypic profiling and interrogation. The generated neurons underwent synaptogenesis and elicit spontaneous Ca(2+) transients. Synaptic vesicle trafficking and release of dopamine in response to depolarizing stimuli was also observed. Under whole-cell current-and-voltage clamp, recordings showed polarized neurons (Vm=-70 mV) and voltage-dependent potassium currents, which included A-type-like currents. Glutamate-induced currents sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate antagonists revealed the existence of functional glutamate receptors. Molecular and phenotypic profiling showed recapitulation of midbrain patterning events, and remodeling toward increased similarity to human brain features, such as extracellular matrix composition and metabolic signature. We have developed a robust and reproducible human 3D neural cell model, which may be extended to patient-derived induced pluripotent stem cells, broadening the applicability of this model.

Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy.

The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

3D cultures of human neural progenitor cells: dopaminergic differentiation and genetic modification. [corrected].

Central nervous system (CNS) disorders remain a formidable challenge for the development of efficient therapies. Cell and gene therapy approaches are promising alternatives that can have a tremendous impact by treating the causes of the disease rather than the symptoms, providing specific targeting and prolonged duration of action. Hampering translation of gene-based therapeutic treatments of neurodegenerative diseases from experimental to clinical gene therapy is the lack of valid and reliable pre-clinical models that can contribute to evaluate feasibility and safety. Herein we describe a robust and reproducible methodology for the generation of 3D in vitro models of the human CNS following a systematic technological approach based on stirred culture systems. We took advantage of human midbrain-derived neural progenitor cells (hmNPCs) capability to differentiate into the various neural phenotypes and of their commitment to the dopaminergic lineage to generate differentiated neurospheres enriched in dopaminergic neurons. Furthermore, we describe a protocol for efficient gene transfer into differentiated neurospheres using CAV-2 viral vectors and stable expression of the transgene for at least 10 days. CAV-2 vectors, derived from canine adenovirus type 2, are promising tools to understand and treat neurodegenerative diseases, in particular Parkinson's disease. CAV-2 vectors preferentially transduce neurons and have an impressive level of axonal retrograde transport in vivo. Our model provides a practical and versatile in vitro approach to study the CNS in a 3D cellular context. With the successful differentiation and subsequent genetic modification of neurospheres we are increasing the collection of tools available for neuroscience research and contributing for the implementation and widespread utilization of 3D cellular CNS models. These can be applied to study neurodegenerative diseases such as Parkinson's disease; to study the interaction of viral vectors of therapeutic potential within human neural cell populations, thus enabling the introduction of specific therapeutic genes for treatment of CNS pathologies; to study the fate and effect of delivered therapeutic genes; to study toxicological effects. Furthermore these methodologies may be extended to other sources of human neural stem cells, such as human pluripotent stem cells, including patient-derived induced pluripotent stem cells.

Gene stage-specific expression in the microenvironment of pediatric myelodysplastic syndromes.

Using cDNA microarray assays we have observed a clear difference in the gene expression pattern between bone marrow stromal cells obtained from healthy children (CT) and from pediatric patients with either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) associated with MDS (MDS-AML). The global gene function profiling analysis indicated that in the pediatric MDS microenvironment the disease stages may be characterized mainly by underexpression of genes associated with biological processes such as transport. Furthermore, a subset of downregulated genes related to endocytosis and protein secretion was able to discriminate MDS from MDS-AML.