Conference Agenda

Brain organoids are in vitro three-dimensional self-organized neural structures, which enable disease modelling and drug screening. However, their use may be limited by their high batch-to-batch variability, long differentiation time (10–20 weeks), and high production costs. To overcome these issues, we recently developed a highly standardized and reproducible murine brain organoid model starting from embryonic neural stem cells. Together with morphological and functional changes, we described a metabolic switch of murine brain organoids from glycolytic to mitochondrial oxidative phosphorylation (OXPHOS) which supports their full neuronal maturation that is accomplished in approximately 30 days in vitro.

Modulation of energy metabolism is emerging as a key aspect associated with cell fate transition; in developing CNS, while undifferentiated stem cells mainly rely on glycolysis, the activation of OXPHOS is a typical signature of the initiation of neurogenesis. Therefore, we took advantage of, and we describe here, the use of murine brain organoids to model neurodevelopmental disorders associated in humans with inborn errors of metabolism, and to test early pharmacological intervention modulating mitochondrial function.

Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide the opportunity to model human nervous system development in vitro since the initial stages of embryogenesis. Moreover, patient-derived iPSCs and gene editing allow generating in vitro models of human nervous system diseases, such as neurodevelopmental and neurodegenerative diseases. A further advancement comes from the possibility to differentiate PSCs as bi-dimensional or three-dimensional constructs resembling organs (organoids) or early embryonic stages (neuruloids and gastruloids). Such "-oid" systems not only contain the variety of the cell types present in vivo, but also mimick to some extent the architecture of their native counterparts. Here we show how organoids can be used to model neurodevelopmental and neurodegenerative diseases, and how neuruloids and gastruloids can be used as platforms for drug testing. Advantages and limitations of such innovative experimental models in neuroscience research will be discussed.

Brain developmental disorders and neurodegenerative diseases represent an area of neuroscience in which the underlying pathogenetic mechanisms are still largely unknown due to the lack of adequate experimental models. Indeed, the main limitation for the study of diseases involving the central nervous system is the lack of a realistic experimental model able to recapitulate the complexity of human living neuronal tissue. Therefore, in recent years many efforts have been made to develop biological systems capable of recapitulating the pathogenesis and distinctive features of these disorders in a physiological-like environment. In this attempt, a considerable contribution derives by the progress in the field of stem cells induced to pluripotency (induced pluripotent derived stem cells, iPSCs) which allow to obtain mature neurons starting from somatic cells (fibroblasts or mononuclear cells of the blood). IPSCs are currently the basis for producing more complex, three-dimensional cell models such as organoids or organ on chip. A great advantage of such three-dimensional development models derives from the fact that with this approach it is possible to mimic the characteristics of the tissue, reproducing the microenvironment and cell-cell connections. In this context, some 3D culture systems used for the expansion of neural and non-neural stem cells will be considered and discussed. Some three-dimensional experimental models for the study of the central nervous system will also be presented. I will try to focus the presentation on the potential applications but also on any critical issues.

Response to stress is a key physiological process finely orchestrated by the brain. However, failure in activating coping strategies may lead to maladaptive consequences in some individuals favoring the onset of pathological conditions, especially psychiatric disorders such as depression. Understanding the mechanisms underlying the trajectories of stress vulnerability/resilience would be crucial to develop new strategical therapeutic approaches. Neuroinflammation has been reported to contribute to the maladaptive consequences of stress by increasing pro-inflammatory cytokines, activating the hypothalamus-pituitary-adrenal (HPA) axis, and affecting neuronal apoptosis and neuroplasticity. Microglia, being the immune cells in the central nervous system, play crucial roles in neuroinflammation. Moreover, microglia can directly model neurons, by engulfing synaptic elements, through a process known as synaptic pruning whose alterations have been associated to psychiatric disorders. Nevertheless, little is known about the role of microglial morphological changes in the response to stress.

In this study, we used high-throughput microscopy and three-dimensional image analysis to investigate brain area-specific changes in microglia morphology and interaction with postsynaptic neuronal spines in vulnerability and resilience to chronic stress.

We applied the chronic mild stress animal model of depression on rats and deemed animals vulnerable or resilient according to the anhedonic-like phenotype measured by the sucrose preference test. After brain collection, we performed immunohistochemistry and deep microscope acquisition on hippocampal and prefrontal cortex coronal sections, followed by high performance image analysis with Imaris. Our results showed an interesting phenotypical heterogeneity of the microglia between hippocampus and prefrontal cortex. Moreover, microglia from resilient and vulnerable rats were characterized by distinct morphological features, especially in the hippocampus, suggesting possible functional consequences. Finally, the number of post-synapses was reduced in the hippocampus of stressed animals with resilient rats showing a significantly higher microglia-mediated engulfment. Overall, our study supports a putative role for microglia remodeling in vulnerability and resiliency to stress.

Peripheral neuropathies are a condition in which peripheral nerves are damaged. This condition affects millions of people every year worldwide and can be caused by external trauma, or by pathologies that impact peripheral nervous system components. Biocompatible scaffolds are emerging as an important tool to promote nerve regeneration in case of resection. However, when there is no physical damage of the nerve an effective drug delivery system is still lacking. To address these issues, we are working on two different approaches.

We have already demonstrated that nano/micro-grooved substrates are capable to direct neuronal and glial cell differentiation, polarization, and migration. We developed microstructured scaffolds, with specific directional topographies and tuneable stiffness, for peripheral nerve regeneration: our scaffolds, made of biodegradable and soft materials (compliant with nerve mechanics), were tested in vitro with neuronal and glial cell models.

Moreover, the restricted permeability of nerves, due to the presence of the blood-nerve barrier (BNB), makes difficult to transport drugs into their structure. Polymeric nanoparticles are under investigation for their ability to pass biological barriers (such as the blood-brain barrier). We developed polymeric nanoparticles (NPs) functionalized with peripheral nerve targeting molecules, procaine and a peptide, NP41. We tested their biocompatibility and internalization capability in vitro in neural cell models. These nanoparticles can be further loaded with drugs of interest.

These two strategies can be either used as stand-alone systems or can be further combined to create innovative devices (e.g. scaffolds functionalized/enriched with NPs loaded with compounds of interest). Such devices would be able to provide both a physical support for regeneration, and a controlled release of drugs for treating a wide variety of pathological conditions impacting PNS (e.g. nerve trauma, tumor resections, neurodegenerative disorders).