Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence <em>In Vitro</em>

Abstract

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated betagalactosidase staining.

Choroid plexus defects in Down syndrome brain organoids enhance neurotropism of SARS-CoV-2

“…We exposed human ES and iPSC lines (fig. S1A) (H9 ( 34 ), WTC ( 35 ), and G22 ( 35 ) to dual SMAD inhibition with SB and LDN for 3 days, which resulted in the efficient generation of hNEct cells that generate neural stem cells characterized by the expression of SOX2 and NESTIN (fig. S1B).…”

“…S1D), we demonstrated that different concentrations of BMP4 altered the content of neural progenitors. Recently, the in vitro generation of hNEct from hPSCs has been instrumental in the production of forebrain cells in conventional monolayer culture and in 3D organoid models ( 35 , 64 , 65 ). Here, we took advantage of the developmental potential of hNEct sheets to generate a complex 3D ChPCO model, in which two different brain domains, cortical neurons and ChP-like epithelium, develop in parallel from common hNEct progenitors that self-organize, and interact to promote the formation of ventricle-like structures.…”

“…Fresh N2 medium with inhibitors was added daily. On the fourth day, induced NEct colonies were lifted using dispase (2.4 U/ml) to form neural spheroids as we recently reported ( 35 ). For the next 4 days, these spheroids were cultured in N2 medium supplemented daily with bFGF (40 ng/ml; R&D Systems, catalog no.…”

Utilizing hiPSC-derived oligodendrocytes to study myelin pathophysiology in neuropsychiatric and neurodegenerative disorders.

“…Thus, directed differentiation typically produces a better representation of the brain region of interest, as it guides NPCs through specific developmental stages to become a particular cell type of interest such as excitatory neurons, interneurons, oligodendrocytes, and/or astrocytes. The majority of available established protocols for generating glial cells result in mixed cultures, composed of neurons, oligodendrocytes, and astrocytes and depending on the protocol the cell type of interest can be generated with a higher ratio to other neural cell types ( Shaker et al, 2022 ). Below we discuss specific guided differentiation protocols that are developed to enrich cultures with oligodendrocytes ( Table 1 and Figure 2 ).…”

A Perspective: Challenges in Dementia Research

“…Model systems that explain only a very few of the changes will not be as valuable as systems that explain many different changes. Models can be based in animals (with caution) [ 99 ], in vitro systems [ 100 ], mathematical or computational [ 101 , 102 ]. These models will be critical to exploring and creating new ideas.…”