Modeling Human Neural Functionality In Vitro : Three-Dimensional Culture for Dopaminergic Differentiation (original) (raw)
Related papers
Methods, 2012
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 (hmNPC) 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.
Generation of hiPSC-Derived Functional Dopaminergic Neurons in Alginate-Based 3D Culture
Frontiers in Cell and Developmental Biology
Human induced pluripotent stem cells (hiPSCs) represent an unlimited cell source for the generation of patient-specific dopaminergic (DA) neurons, overcoming the hurdle of restricted accessibility to disease-affected tissue for mechanistic studies on Parkinson’s disease (PD). However, the complexity of the human brain is not fully recapitulated by existing monolayer culture methods. Neurons differentiated in a three dimensional (3D) in vitro culture system might better mimic the in vivo cellular environment for basic mechanistic studies and represent better predictors of drug responses in vivo. In this work we established a new in vitro cell culture system based on the microencapsulation of hiPSCs in small alginate/fibronectin beads and their differentiation to DA neurons. Optimization of hydrogel matrix concentrations and composition allowed a high viability of embedded hiPSCs. Neural differentiation competence and efficiency of DA neuronal generation were increased in the 3D cultu...
BMC Proceedings, 2011
The development of new drugs for human Central Nervous System (CNS) diseases has traditionally relied on 2D in vitro cell models and genetically engineered animal models. However, those models often diverge considerably from that of human phenotype (anatomical, developmental and biochemical differences) [1] contributing to a high attrition rate -only 8% of CNS drugs entering clinical trials end up being approved . Human 3D in vitro models are useful complementary tools towards more accurate evaluation of drug candidates in pre-clinical stages, as they present an intermediate degree of complexity in terms of cell-cell and cellmatrix interactions, between the traditional 2D monolayer culture conditions and the complex brain and can be a better starting point for the analysis of the in vivo context. Aiming at developing novel 3D in vitro models of the CNS, this work focus on the implementation of long-term cultures of human midbrain-derived neural stem cells (hmNSC) for the scalable supply of neuralsubtype cells, with a focus on the dopaminergic lineage, following a systematic technological approach based on stirred culture systems.
Advanced Science
nature of the disease in conventional in vitro models and the excessive reliance on animal models partly explain the disappointingly high failure rate of new candidate molecules in clinical trials. [1,2] New technological advancements have failed to translate into successful curative pharmacological options and no definitive diseasemodifying therapy is currently available. In this scenario, human induced pluripotent stem cells (iPSCs) represent a promising tool for the generation of relevant human in vitro models, due to their ability to differentiate into any cell type of the body. [3] However, the use of iPSC technology alone is often not sufficient to account for all the limitations of modeling complex diseases in a dish. Standard 2D cell culture systems do not offer an ideal setup to study highly ramified cells such as neurons. In 2D cultures, the dendrites and growth cones are unrealistically flattened, limiting the acquisition of full cellular functionality, and the cellular microenvironment is poorly modelled. It has been shown that cells in a 3D in vitro setting are subjected to mechanostructural cues which bring them closer to physiological conditions. [4] Surrounded by matrix surrogates, cells experience a more physiological equilibration and transport of soluble factors. [4,5] Notably, several groups have reported significantly different gene and protein expression profiles when comparing 2D and 3D cultures. [6] Cells cultured in 2D showed ≈30% of differentially expressed genes compared to cells in vivo. [7] It is Parkinson's disease (PD)-specific neurons, grown in standard 2D cultures, typically only display weak endophenotypes. The cultivation of PD patientspecific neurons, derived from induced pluripotent stem cells carrying the LRRK2-G2019S mutation, is optimized in 3D microfluidics. The automated image analysis algorithms are implemented to enable pharmacophenomics in disease-relevant conditions. In contrast to 2D cultures, this 3D approach reveals robust endophenotypes. High-content imaging data show decreased dopaminergic differentiation and branching complexity, altered mitochondrial morphology, and increased cell death in LRRK2-G2019S neurons compared to isogenic lines without using stressor agents. Treatment with the LRRK2 inhibitor 2 (Inh2) rescues LRRK2-G2019S-dependent dopaminergic phenotypes. Strikingly, a holistic analysis of all studied features shows that the genetic background of the PD patients, and not the LRRK2-G2019S mutation, constitutes the strongest contribution to the phenotypes. These data support the use of advanced in vitro models for future patient stratification and personalized drug development. Disease Modeling
Frontiers in Cell and Developmental Biology, 2020
Neurons derived from human induced pluripotent stem cells (hiPSC-derived neurons) offer novel opportunities for the development of preclinical models of human neurodegenerative diseases (NDDs). Recent advances in the past few years have increased substantially the potential of these techniques and have uncovered new challenges that the field is facing. Here, we outline and discuss challenges related to the functional characterization of hiPSC-derived neurons and propose ways to overcome current difficulties. In particular, the enormous variability among studies in the electrical properties of hiPSC-derived neurons and broad differences in cell maturation are factors that impair reproducibility. Furthermore, we discuss how the use of 3D brain organoids are of help in resolving some difficulties posed by 2D cultures. Finally, we elaborate on recent and future advances that may help to overcome the discussed challenges and speed-up progress in the field.
Micro Three-Dimensional Neuronal Cultures Generate Developing Cortex-Like Activity Patterns
Frontiers in Neuroscience, 2020
Studies aimed at neurological drug discovery have been carried out both in vitro and in vivo. In vitro cell culture models have showed potential as drug testing platforms characterized by high throughput, low cost, good reproducibility and ease of handling and observation. However, in vitro neuronal culture models are facing challenges in replicating in vivo-like activity patterns. This work reports an in vitro culture technique that is capable of producing micro three-dimensional (µ3D) cultures of only a few tens of neurons. The µ3D cultures generated by this method were uniform in size and density of neurons. These µ3D cultures had complex spontaneous synchronized neuronal activity patterns which were similar to those observed in the developing cortex and in much larger 3D cultures, but not in 2D cultures. Bursts could be reliably evoked by stimulation of single neurons. Synchronized bursts in µ3D cultures were abolished by inhibitors of glutamate receptors, while inhibitors of GABA A receptors had a more complex effect. This pharmacological profile is similar to bursts in neonatal cortex. Since large numbers of reproducible µ3D cultures can be created and observed in parallel, this model of the developing cortex may find applications in high-throughput drug discovery experiments.
Scientific Reports, 2014
Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These networks are usually grown onto specific rigid substrates (also with embedded electrodes) and lack of most of the constituents of the in-vivo like environment: cell morphology, cell-to-cell interaction and neuritic outgrowth in all directions. Cells in a brain region develop in a 3D space and interact with a complex multi-cellular environment and extracellular matrix. Under this perspective, 3D networks coupled to micro-transducer arrays, represent a new and powerful in-vitro model capable of better emulating in-vivo physiology. In this work, we present a new experimental paradigm constituted by 3D hippocampal networks coupled to Micro-Electrode-Arrays (MEAs) and we show how the features of the recorded network dynamics differ from the corresponding 2D network model. Further development of the proposed 3D in-vitro model by adding embedded functionalized scaffolds might open new prospects for manipulating, stimulating and recording the neuronal activity to elucidate neurophysiological mechanisms and to design bio-hybrid microsystems. S everal studies have been devoted to the introduction of in-vitro 3D neuronal systems but the use of such experimental models is still limited and, as far as we know, no attempt of functional multisite electrophysiological measurements of 3D neuronal networks has been presented in the literature. The potential advantages of 3D engineered constructs are evident as they can be used as a more accurate investigational in-vitro platform or as the basis for developing living bio-hybrid neuro-electronic microsystems in-vitro or in-vivo 1 . Thus the design and implementation of 3D engineered neuronal networks with embedded sensors and recordingstimulating electrodes, would give new opportunities for investigations and applications in the neuroscientific domain. On the other hand, the development of such 3D network architectures and the possibility of chronic and functional electrophysiological recordings pose new challenges in terms of integration between scaffolds and recording-stimulating devices, long-term cell survival, exchange of nutrients, cell coupling with micro-electrodes and micro-sensors, etc. Till now, most of the efforts have been devoted to the development of new materials 2 , passive 3,4 and active scaffolds 5 , and new experimental procedures to guarantee the development of 3D cultured networks; however, multi-site electrophysiological recordings in such 3D neuronal preparations are still lacking.
Modelling neurodegenerative diseases in vitro: Recent advances in 3D iPSC technologies
AIMS Cell and Tissue Engineering, 2018
The discovery of induced pluripotent stem cells (iPSC) 12 years ago has fostered the development of innovative patient-derived in vitro models for better understanding of disease mechanisms. This is particularly relevant to neurodegenerative diseases, where availability of live human brain tissue for research is limited and post-mortem interval changes influence readouts from autopsy-derived human tissue. Hundreds of iPSC lines have now been prepared and banked, thanks to several large scale initiatives and cell banks. Patient-or engineered iPSC-derived neural models are now being used to recapitulate cellular and molecular aspects of a variety of neurodegenerative diseases, including early and pre-clinical disease stages. The broad relevance of these models derives from the availability of a variety of differentiation protocols to generate disease-specific cell types and the manipulation to either introduce or correct disease-relevant genetic modifications. Moreover, the use of chemical and physical three-dimensional (3D) matrices improves control over the extracellular environment and cellular organization of the models. These iPSC-derived neural models can be utilised to identify target proteins and, importantly, provide high-throughput screening for drug discovery. Choosing Alzheimer's disease (AD) as an example, this review describes 3D iPSC-derived neural models and their advantages and limitations. There is now a requirement to fully characterise and validate these 3D iPSC-derived neural models as a viable research tool that is capable of complementing animal models of neurodegeneration and live human brain tissue. With further optimization of differentiation, maturation and aging protocols, as well as the 3D cellular organisation and extracellular matrix to recapitulate more closely, the molecular
Modelling neurodegenerative diseases in vitro: Recent advances in 3D iPSC technologies
AIMS Cell and Tissue Engineering
The discovery of induced pluripotent stem cells (iPSC) 12 years ago has fostered the development of innovative patient-derived in vitro models for better understanding of disease mechanisms. This is particularly relevant to neurodegenerative diseases, where availability of live human brain tissue for research is limited and post-mortem interval changes influence readouts from autopsy-derived human tissue. Hundreds of iPSC lines have now been prepared and banked, thanks to several large scale initiatives and cell banks. Patient-or engineered iPSC-derived neural models are now being used to recapitulate cellular and molecular aspects of a variety of neurodegenerative diseases, including early and pre-clinical disease stages. The broad relevance of these models derives from the availability of a variety of differentiation protocols to generate disease-specific cell types and the manipulation to either introduce or correct disease-relevant genetic modifications. Moreover, the use of chemical and physical three-dimensional (3D) matrices improves control over the extracellular environment and cellular organization of the models. These iPSC-derived neural models can be utilised to identify target proteins and, importantly, provide high-throughput screening for drug discovery. Choosing Alzheimer's disease (AD) as an example, this review describes 3D iPSC-derived neural models and their advantages and limitations. There is now a requirement to fully characterise and validate these 3D iPSC-derived neural models as a viable research tool that is capable of complementing animal models of neurodegeneration and live human brain tissue. With further optimization of differentiation, maturation and aging protocols, as well as the 3D cellular organisation and extracellular matrix to recapitulate more closely, the molecular
Three-dimensional neural constructs: a novel platform for neurophysiological investigation
Journal of Neural Engineering, 2008
Morphological and electrophysiological properties of neural cells are substantially influenced by their immediate extracellular surroundings, yet the features of this environment are difficult to mimic in vitro. Therefore, there is a tremendous need to develop a new generation of culture systems that more closely model the complexity of nervous tissue. To this end, we engineered novel electrophysiologically active 3D neural constructs composed of neurons and astrocytes within a bioactive extracellular matrix-based scaffold. Neurons within these constructs exhibited extensive 3D neurite outgrowth, expressed mature neuron-specific cytoskeletal proteins, and remained viable for several weeks. Moreover, neurons assumed complex 3D morphologies with rich neurite arborization and clear indications of network connectivity, including synaptic junctures. Furthermore, we modified whole-cell patch clamp techniques to permit electrophysiological probing of neurons deep within the 3D constructs, revealing that these neurons displayed both spontaneous and evoked electrophysiological action potentials and exhibited functional synapse formation and network properties. This is the first report of individual patch clamp recordings of neurons deep within 3D scaffolds. These tissue engineered cellular constructs provide an innovative platform for neurobiological and electrophysiological investigations, serving as an important step towards the development of more physiologically relevant neural tissue models.