Genetic Modification of Brain Organoids

Abstract

Brain organoids have become increasingly used systems allowing 3D-modeling of human brain development, evolution, and disease. To be able to make full use of these modeling systems, researchers have developed a growing toolkit of genetic modification techniques. These techniques can be applied to mature brain organoids or to the preceding embryoid bodies (EBs) and founding cells. This review will describe techniques used for transient and stable genetic modification of brain organoids and discuss their current use and respective advantages and disadvantages. Transient approaches include adeno-associated virus (AAV) and electroporation-based techniques, whereas stable genetic modification approaches make use of lentivirus (including viral stamping), transposon and CRISPR/Cas9 systems. Finally, an outlook as to likely future developments and applications regarding genetic modifications of brain organoids will be presented.

Neurobiology of cannabinoid receptor signaling

“…Furthermore, SNPs in ECS components 11 can also be investigated using human organoids generated from induced pluripotent stem cells, 184,185 with the additional potential of genomic manipulation using CRISPR/Cas technology 186 and pharmacological interventions, for example, with cannabinoids, 187 together with state-of-the-art analyses, such as single-cell RNA-seq. 188 These approaches will further strengthen mechanistic insights into the roles of the ECS in psychiatric disorders.…”

Electrophysiological Analysis of Brain Organoids: Current Approaches and Advancements

“…Improved genetic technologies and transcriptomics, such as single-cell RNA sequencing, have allowed for detailed characterization of cell types and developmental states within organoids ( Quadrato et al, 2017 ; Fischer et al, 2019 ); however, functional analysis of brain organoids is limited ( Schröter et al, 2018 ; Poli et al, 2019 ). Classical electrophysiological methods such as patch clamp allow for high temporal resolution of neural activity in organoids but offer little spatial resolution for assessment of whole-organoid activity ( Pasca et al, 2015 ).…”

Human-specificARHGAP11Bis necessary and sufficient for human-type basal progenitor levels in primate brain organoids

“…Thanks to the seminal work of a few laboratories (Kadoshima et al, 2013;Karzbrun et al, 2018;Lancaster et al, 2013;Pasca et al, 2015;Qian et al, 2016;Quadrato et al, 2017), the brain organoid technology provides a way out of this dilemma. A specific subtype of brain organoids, the cerebral organoids, are relatively small (a few mm in diameter) three-dimensional (3D) structured cell assemblies that can be grown from embryonic stem cells (ESCs) (in the case of human) or induced pluripotent stem cells (iPSCs) (in the case of human and chimpanzee) and that emulate cerebral tissue (Arlotta, 2018;Di Lullo & Kriegstein, 2017;Fischer et al, 2019;Heide et al, 2018;Kelava & Lancaster, 2016;Lancaster et al, 2013). Cerebral organoids have been shown to exhibit several (albeit not all) of the hallmarks of developing neocortical tissue, including the two principal germinal zones, the ventricular zone (VZ) and the subventricular zone (SVZ), as well as the two major classes of progenitor cells therein, the apical progenitors (APs) and the basal progenitors (BPs) (Heide et al, 2018;Kadoshima et al, 2013;Lancaster et al, 2013;Qian et al, 2016;Quadrato et al, 2017).…”

“…These and other studies (Kanton et al, 2019;Pollen et al, 2019) have established that the intrinsic behaviour of cNPCs and the neuron generation therefrom as observed in human vs. chimpanzee cerebral organoids allows the identification of human-specific features of neocortical development. However, cerebral organoids also offer the opportunity of extrinsic genetic manipulation (Fischer et al, 2019). This is particularly relevant in the case of humanspecific genes that in developing neocortex are preferentially expressed in cNPCs and hence have been implicated in human-specific features of neocortical development (Fiddes et al, 2018;Florio et al, 2015;Florio et al, 2018;Suzuki et al, 2018).…”

“…exhibiting numerous ventricular structures (Figure 1-figure supplement 1A). Various mixtures of DNA constructs, consisting of a cytosolic-GFP expression vector and either an expression vectorwith the cDNA of interest or the corresponding control vector, were then microinjected into the lumen of the larger ventricle-like structures within the cerebral organoids, followed by electroporation to transfect the cNPCs in the VZ(Figure 1-figure supplement 1A,(Fischer et al, 2019;Lancaster et al, 2013;Li et al, 2017)). Depending on the specific scientific question asked, cerebral organoids were fixed 2-10 days after electroporation, in the case of 2 days with addition of BrdU 1 h prior to fixation as indicated(Figure 1-figure supplement 1A).…”