Abstract
Human cerebral organoid (CO) is a three-dimensional (3D) cell culture system that recapitulates the developing human brain. While CO has proved an invaluable tool for studying neurological disorders in a more clinically relevant matter, there have still been several shortcomings including CO variability and reproducibility as well as lack of or underrepresentation of certain cell types typically found in the brain. As the technology to generate COs has continued to improve, more efficient and streamlined protocols have addressed some of these issues. Here we present a novel scalable and simplified system to generate microglia-containing CO (MCO). We characterize the cell types and dynamic development of MCOs and validate that these MCOs harbor microglia, astrocytes, neurons, and neural stem/progenitor cells, maturing in a manner that reflects human brain development. We introduce a novel technique for the generation of embryoid bodies (EBs) directly from induced pluripotent stem cells (iPSCs) that involves simplified steps of transitioning directly from 3D cultures as well as orbital shaking culture in a standard 6-well culture plate. This allows for the generation of MCOs with an easy-to-use system that is affordable and accessible by any general lab.
Highlights
The brain is a complex organ consisting of intricate neural networks with extensive functions
One study modified neural induction condition by reducing heparin concentration, rendering the innate generation of mesodermal cells, which readily differentiated into microglial cells within the cerebral organoid (CO), named Microglia-Containing Cerebral Organoids (MCOs) (Ormel et al, 2018). Following this approach for inclusion of innate microglia that plays an essential role in brain development, neural innate immunity, neuroinflammation, and neurotropic viral infection, we developed a novel scalable and simplified protocol for the generation of MCO from human induced pluripotent stem cell (iPSC)
To generate iPSCs, human fibroblasts from apparently healthy individuals were sent to the University of Pennsylvania iPSC Core for reprogramming using Sendai virus technology (Figure 1A) in mouse embryonic fibroblast (MEF)-feeder culture (Figure 1B)
Summary
The brain is a complex organ consisting of intricate neural networks with extensive functions. These applications include neurodevelopmental disorders such as autism spectrum disorders and related disorders (Mariani et al, 2015; Chailangkarn et al, 2016; Birey et al, 2017; Wang et al, 2017; Mellios et al, 2018; Gouder et al, 2019; Sun et al, 2019), neuropsychiatric disorders such as schizophrenia (Srikanth et al, 2015; Stachowiak et al, 2017; Ye et al, 2017; De Vrij et al, 2019; Kathuria et al, 2020), structural and migration disorders such as microencephaly and lissencephaly (Lancaster et al, 2013; Bershteyn et al, 2017; Iefremova et al, 2017; Li et al, 2017a,b; Fiddes et al, 2018; Karzbrun et al, 2018; Zhang et al, 2019), and even neurotropic viral infections such as Zika Virus (Cugola et al, 2016; Dang et al, 2016; Qian et al, 2016; Gabriel et al, 2017; Li et al, 2017b; Watanabe et al, 2017) and SARSCoV-2 (Jacob et al, 2020; Pellegrini et al, 2020a; Ramani et al, 2020; Yang et al, 2020; Zhang et al, 2020)
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