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Developmental Biology

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

Published: March 24th, 2023



1German Primate Center, Leibniz Institute for Primate Research, 2Max Planck Institute of Molecular Cell Biology and Genetics

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.

As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Investigating the (patho)physiological development and evolution of the cerebral cortex is a formidable task that is hampered by the lack of suitable model systems. Previously, such studies were confined to two-dimensional cell culture models (such as primary neural progenitor or neuronal cell cultures) and evolutionarily distant animal models (such as rodents)1,2. While these models are useful for addressing certain questions, they are limited in modeling the complexity, cell type composition, cellular architecture, and gene expression patterns of the developing human neocortex in healthy and diseased states.....

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1. Culture of primate iPSCs

NOTE: Due to its robustness, the method presented here can be applied to any primate iPSC line. In this article, we describe cerebral organoid production from human (iLonza2.2)29, chimpanzee (Sandra A)30, rhesus macaque (iRh33.1)29, and common marmoset (cj_160419_5)31 iPSC lines. The culture conditions are summarized in Table 1. See th.......

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The protocol described here allows the efficient generation of cerebral organoids from human, chimpanzee, rhesus macaque, and common marmoset iPSC lines with minimal timing alterations required between species (Figure 1A). These organoids can be electroporated in the range of 20 dps to 50 dps, depending on the accessibility of the ventricle-like structures and the abundance of the cell population(s) of interest. However, prior to electroporation, it is important to determine whether the cere.......

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The procedures described here represent a unified protocol for the generation of cerebral organoids from different primate species with a targeted electroporation approach. This allows the ectopic expression of a GOI in a model system that emulates primate (including human) (patho)physiological neocortex development. This unified protocol for the generation of primate cerebral organoids uses the same materials (e.g., media) and protocol steps for all four primate species presented. Developmental differences between these.......

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We apologize to all the researchers whose work could not be cited due to space limitations. We thank Ulrich Bleyer of the technical services at DPZ and Hartmut Wolf of the workshop at MPI-CBG for the construction of the Petri dish electrode chambers; Stoyan Petkov and Rüdiger Behr for providing human (iLonza2.2), rhesus macaque (iRh33.1) and marmoset (cj_160419_5) iPSCs; Sabrina Heide for the cryosectioning and immunofluorescence staining; and Neringa Liutikaite and César Mateo Bastidas Betancourt for critically reading the manuscript. Work in the laboratory of W.B.H. was supported by an ERA-NET NEURON (MicroKin) grant. Work in the laboratory of M.H. was sup....

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Name Company Catalog Number Comments
20 µL Microloader Eppendorf 5242956003
2-Mercaptoethanol Merck 8.05740.0005
35 mm cell culture dishes Sarstedt 83.3900
60 mm cell culture dishes CytoOne CC7682-3359
Activin A Sigma-Aldrich SRP3003
AOC1 Selleckchem S7217
Axio Observer.Z1 Inverted Fluorescence Microscope Zeiss replacable by comparable fluorescent microscopes
AZD0530 Selleckchem  S1006
B-27 Supplement with Vitamin A (retinoic acid, RA) (50x) Gibco 17504-044
B-27 Supplement without Vitamin A (50x) Gibco 12587-010
BTX ECM 830 Square Wave Electroporation System BTX 45-2052
CGP77675 Sigma-Aldrich SML0314
Chimpanzee induced pluripotent stem cell line Sandra A doi: 10.7554/elife.18683 
Common marmoset induced pluripotent stem cell line cj_160419_5 doi: 10.3390/cells9112422
Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) Gibco 11320-033
Dulbecco's phosphate-buffered saline (DPBS) Gibco 14190-094 pH 7.0−7.3; warm to room temperature before use
Fast Green Sigma-Aldrich F7252-5G
Forskolin Selleckchem 2449
GlutaMAX Supplement (100x) Gibco 35050-061 glutamine substitute supplement
Heparin (1 mg/mL stock) Sigma-Aldrich H3149
Human induced pluripotent stem cell line iLonza2.2 doi: 10.3390/cells9061349
Human Neurotrophin-3 (NT-3) PeproTech 450-03
Insulin Sigma-Aldrich 19278
IWR1 Sigma-Aldrich I0161
Leica MS5 stereomicroscope (MDG 17 transmitted-light base) Leica 10473849 replacable by comparable stereomicroscopes
Matrigel Corning 354277/354234 basement membrane matrix; alternatively, Geltrex (ThermoFisher Scientific, A1413302) can be used
MEM Non-Essential Amino Acids Solution (100x) Sigma-Aldrich M7145
N-2 Supplement (100x) Gibco 17502-048
Neurobasal medium Gibco 21103-049
Parafilm Sigma-Aldrich P7793
Paraformaldehyde  Merck 818715 handle with causion due to cancerogenecity
Penicillin/Streptomycin (10,000 U/mL) PanBiotech P06-07100
Petri dish electrode chamber self-produced (see Supplemental File 1) also commertially available
Pre-Pulled Glass Pipettes WPI TIP10LT borosilicate glass pipettes with long taper, 10 µm tip diameter
Pro-Survival Compound MerckMillipore 529659
Recombinant Human/Murine/RatBrain-Derived Neurotrophic Factor (BDNF) PeproTech AF-450-02
Rhesus macaque induced pluripotent stem cell line iRh33.1 doi: 10.3390/cells9061349
StemMACS iPS-Brew XF Miltenyi Biotech 130-104-368
StemPro Accutase Cell Dissociation Reagent Gibco A1110501 proteolytic and collagenolytic enzyme mixture
TrypLE Gibco 12604-013 recombinant trypsin substitute; warm to room temperature before use
Ultra-Low Attachment 96-well plates Costar 7007
Y27632 Stemcell Technologies 72305

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