Conference Dates

May 6-11, 2018

Abstract

Brain microenvironment plays an important role in neurodevelopment and pathology, where extracellular matrix (ECM) and soluble factors modulate multiple cellular processes. Neural cell culture typically relies on the use of heterologous matrices that poorly resemble the brain ECM or reflect its pathological features. We have previously demonstrated that perfusion stirred-tank bioreactor-based 3D differentiation of human neural stem cells (NSC) - pSTR-neurospheroids, sustains the concomitant differentiation of the three neural cell lineages (neurons, astrocytes and oligodendrocytes) and the establishment of physiologically relevant cell-cell interactions. Here, we hypothesized that if the pSTR-neurospheroid strategy would also allow the deposition of native neural ECM components and diffusion of secreted factors, it would be possible to: (i) mimic the cellular and microenvironment remodeling occurring during neural differentiation without the confounding effects of exogenous matrices; (ii) recapitulate the pathological phenotypes of diseases in which alteration of homotypic and heterotypic cell-cell interactions and ECM components are relevant. To demonstrate the first point, we analyzed pSTR-neurospheroid differentiation by quantitative transcriptome (NGS) and proteome (SWATH-MS). Data showed that neurogenic developmental pathways were recapitulated, with significant changes at cell membrane and ECM composition, diverging from the 2D differentiation profile. A significant enrichment in structural proteoglycans typical of brain ECM, along with downregulation of basement membrane constituents was observed. Moreover, higher expression of synaptic and ion transport machinery in pSTR-neurospheroids suggest higher neuronal maturation than in 2D. Having shown recapitulation of neural microenvironmental dynamics in pSTR-neurospheroids, we used Mucopolysaccharidosis VII (MPSVII) as a disease case study. MPS VII is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity, which leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. In pSTR-neurospheroids generated from hiPSC of a MPS VII patient, the main molecular disease hallmarks were recapitulated, namely accumulation of GAGs. Notably, MPS VII neurospheroids showed reduced neuronal activity and a disturbance in network functionality, with alterations both in connectivity and synchronization, not observed in 2D cultures. These data provide insight into the interplay between reduced β-gluc activity, GAG accumulation, alterations in the neural network, and its impact on MPS VII-associated cognitive defects. Overall we demonstrate that neural cellular and extracellular developmental and pathological features are recapitulated in healthy and diseased pSTR-neurospheroids, respectively. These can be valuable in vitro models to address molecular defects associated with neurological disorders that affect neural microenvironment homeostasis. Moreover, the 3D neuronal connectivity assay developed is a new tool with potential to assess other lysosomal storage diseases and neurodegenerative diseases that have variable phenotypes. Acknowledgements: SFRH/BD/78308/2011, SFRH/BD/52202/2013 and SFRH/BD/52473/2014 PhD fellowships from FCT, Portugal and iNOVA4Health-UID/Multi/04462/2013, supported by FCT/ MEC, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement.

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