Hippocampal Neuronal Culture v1

This protocol describes the procedure for hippocampal neuronal cultures from new - born mouse pups.

Tuning Physicochemical Properties of a Macroporous Polysaccharide-Based Scaffold for 3D Neuronal Culture

Central nervous system (CNS) lesions are a leading cause of death and disability worldwide. Three-dimensional neural cultures in biomaterials offer more physiologically relevant models for disease studies, toxicity screenings or in vivo transplantations. Herein, we describe the development and use of pullulan/dextran polysaccharide-based scaffolds for 3D neuronal culture. We first assessed scaffolding properties upon variation of the concentration (1%, 1.5%, 3% w/w) of the cross-linking agent, sodium trimetaphosphate (STMP). The lower STMP concentration (1%) allowed us to generate scaffolds with higher porosity (59.9 ± 4.6%), faster degradation rate (5.11 ± 0.14 mg/min) and lower elastic modulus (384 ± 26 Pa) compared with 3% STMP scaffolds (47 ± 2.1%, 1.39 ± 0.03 mg/min, 916 ± 44 Pa, respectively). Using primary cultures of embryonic neurons from PGKCre, Rosa26tdTomato embryos, we observed that in 3D culture, embryonic neurons remained in aggregates within the scaffolds and did not attach, spread or differentiate. To enhance neuronal adhesion and neurite outgrowth, we then functionalized the 1% STMP scaffolds with laminin. We found that treatment of the scaffold with a 100 μg/mL solution of laminin, combined with a subsequent freeze-drying step, created a laminin mesh network that significantly enhanced embryonic neuron adhesion, neurite outgrowth and survival. Such scaffold therefore constitutes a promising neuron-compatible and biodegradable biomaterial.

Induced Pluripotent Stem Cell-Derived Dopaminergic Neurons from Familial Parkinson’s Disease Patients Display α-Synuclein Pathology and Abnormal Mitochondrial Morphology

Accumulation of α-synuclein (α-syn) into Lewy bodies (LBs) and mitochondrial abnormalities are the two cardinal pathobiological features of Parkinson’s disease (PD), which are associated with the loss of dopaminergic neurons. Although α-syn accumulates in many different cellular and mouse models, these models generally lack LB features. Here, we generated midbrain dopaminergic (mDA) neuronal cultures from induced pluripotent stem cells (iPSCs) derived from familial PD (fPD) patients and healthy controls. We show that mDA neuronal cultures from fPD patients with A53T mutation and α-syn gene (SNCA) triplication display pathological α-syn deposits, which spatially and morphologically resemble LBs. Importantly, we did not find any apparent accumulation of pathological α-syn in mDA neuronal culture derived from a healthy donor. Furthermore, we show that there are morphological abnormalities in the mitochondrial network in mDA neuronal cultures from fPD patients. Consequently, these cells were more susceptible to mitochondrial damage compared with healthy donor-derived mDA neuronal cultures. Our results indicate that the iPSC-derived mDA neuronal culture platform can be used to investigate the spatiotemporal appearance of LBs, as well as their composition, architecture, and relationship with mitochondrial abnormalities.

Improved Method for Efficient Generation of Functional Neurons from Murine Neural Progenitor Cells

Neuronal culture was used to investigate neuronal function in physiological and pathological conditions. Despite its inevitability, primary neuronal culture remained a gold standard method that requires laborious preparation, intensive training, and animal resources. To circumvent the shortfalls of primary neuronal preparations and efficiently give rise to functional neurons, we combine a neural stem cell culture method with a direct cell type-conversion approach. The lucidity of this method enables the efficient preparation of functional neurons from mouse neural progenitor cells on demand. We demonstrate that induced neurons (NPC-iNs) by this method make synaptic connections, elicit neuronal activity-dependent cellular responses, and develop functional neuronal networks. This method will provide a concise platform for functional neuronal assessments. This indeed offers a perspective for using these characterized neuronal networks for investigating plasticity mechanisms, drug screening assays, and probing the molecular and biophysical basis of neurodevelopmental and neurodegenerative diseases.

Review: In vitro Cell Platform for Understanding Developmental Toxicity

Developmental toxicity and its affiliation to long-term health, particularly neurodegenerative disease (ND) has attracted significant attentions in recent years. There is, however, a significant gap in current models to track longitudinal changes arising from developmental toxicity. The advent of induced pluripotent stem cell (iPSC) derived neuronal culture has allowed for more complex and functionally active in vitro neuronal models. Coupled with recent progress in the detection of ND biomarkers, we are equipped with promising new tools to understand neurotoxicity arising from developmental exposure. This review provides a brief overview of current progress in neuronal culture derived from iPSC and in ND markers.