JoVE Logo
Faculty Resource Center

Sign In





Representative Results





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.....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
20 µL MicroloaderEppendorf5242956003
35 mm cell culture dishesSarstedt83.3900
60 mm cell culture dishesCytoOneCC7682-3359
Activin ASigma-AldrichSRP3003
Axio Observer.Z1 Inverted Fluorescence MicroscopeZeissreplacable by comparable fluorescent microscopes
AZD0530Selleckchem S1006
B-27 Supplement with Vitamin A (retinoic acid, RA) (50x)Gibco17504-044
B-27 Supplement without Vitamin A (50x)Gibco12587-010
BTX ECM 830 Square Wave Electroporation SystemBTX45-2052
Chimpanzee induced pluripotent stem cell line Sandra Adoi: 10.7554/elife.18683 
Common marmoset induced pluripotent stem cell line cj_160419_5doi: 10.3390/cells9112422
Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)Gibco11320-033
Dulbecco's phosphate-buffered saline (DPBS)Gibco14190-094pH 7.0−7.3; warm to room temperature before use
Fast GreenSigma-AldrichF7252-5G
GlutaMAX Supplement (100x)Gibco35050-061glutamine substitute supplement
Heparin (1 mg/mL stock)Sigma-AldrichH3149
Human induced pluripotent stem cell line iLonza2.2doi: 10.3390/cells9061349
Human Neurotrophin-3 (NT-3)PeproTech450-03
Leica MS5 stereomicroscope (MDG 17 transmitted-light base)Leica10473849replacable by comparable stereomicroscopes
MatrigelCorning354277/354234basement membrane matrix; alternatively, Geltrex (ThermoFisher Scientific, A1413302) can be used
MEM Non-Essential Amino Acids Solution (100x)Sigma-AldrichM7145
N-2 Supplement (100x)Gibco17502-048
Neurobasal mediumGibco21103-049
Paraformaldehyde Merck818715handle with causion due to cancerogenecity
Penicillin/Streptomycin (10,000 U/mL)PanBiotechP06-07100
Petri dish electrode chamberself-produced (see Supplemental File 1)also commertially available
Pre-Pulled Glass PipettesWPITIP10LTborosilicate glass pipettes with long taper, 10 µm tip diameter
Pro-Survival CompoundMerckMillipore529659
Recombinant Human/Murine/RatBrain-Derived Neurotrophic Factor (BDNF)PeproTechAF-450-02
Rhesus macaque induced pluripotent stem cell line iRh33.1doi: 10.3390/cells9061349
StemMACS iPS-Brew XFMiltenyi Biotech130-104-368
StemPro Accutase Cell Dissociation ReagentGibcoA1110501proteolytic and collagenolytic enzyme mixture
TrypLEGibco12604-013recombinant trypsin substitute; warm to room temperature before use
Ultra-Low Attachment 96-well platesCostar7007
Y27632Stemcell Technologies72305

  1. Marchetto, M. C. N., Winner, B., Gage, F. H. Pluripotent stem cells in neurodegenerative and neurodevelopmental diseases. Human Molecular Genetics. 19 (R1), R71-R76 (2010).
  2. Zhao, X., Bhattacharyya, A. Human models are needed for studying human neurodevelopmental disorders. The American Journal of Human Genetics. 103 (6), 829-857 (2018).
  3. Zhang, W., et al. Modeling microcephaly with cerebral organoids reveals a WDR62–CEP170–KIF2A pathway promoting cilium disassembly in neural progenitors. Nature Communications. 10 (1), 2612 (2019).
  4. Sasaki, E., et al. Generation of transgenic non-human primates with germline transmission. Nature. 459 (7246), 523-527 (2009).
  5. Niu, Y., et al. Transgenic rhesus monkeys produced by gene transfer into early-cleavage–stage embryos using a simian immunodeficiency virus-based vector. Proceedings of the National Academy of Sciences of the United States of America. 107 (41), 17663-17667 (2010).
  6. Niu, Y., et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 156 (4), 836-843 (2014).
  7. Shi, L., et al. Transgenic rhesus monkeys carrying the human MCPH1 gene copies show human-like neoteny of brain development. National Science Review. 6 (3), 480-493 (2019).
  8. Heide, M., et al. Human-specific ARHGAP11B increases size and folding of primate neocortex in the fetal marmoset. Science. 369 (6503), 546-550 (2020).
  9. Kadoshima, T., et al. Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell–derived neocortex. Proceedings of the National Academy of Sciences of the United States of America. 110 (50), 20284-20289 (2013).
  10. Lancaster, M. A., et al. Cerebral organoids model human brain development and microcephaly. Nature. 501 (7467), 373-379 (2013).
  11. Kelava, I., Lancaster, M. A. Stem cell models of human brain development. Cell Stem Cell. 18 (6), 736-748 (2016).
  12. Lullo, E. D., Kriegstein, A. R. The use of brain organoids to investigate neural development and disease. Nature Reviews Neuroscience. 18 (10), 573-584 (2017).
  13. Arlotta, P. Organoids required! A new path to understanding human brain development and disease. Nature Methods. 15 (1), 27-29 (2018).
  14. Heide, M., Huttner, W. B., Mora-Bermúdez, F. Brain organoids as models to study human neocortex development and evolution. Current Opinion in Cell Biology. 55, 8-16 (2018).
  15. Qian, X., Song, H., Ming, G. Brain organoids: Advances, applications and challenges. Development. 146 (8), dev166074 (2019).
  16. Sun, N., Meng, X., Liu, Y., Song, D., Jiang, C., Cai, J. Applications of brain organoids in neurodevelopment and neurological diseases. Journal of Biomedical Science. 28 (1), 30 (2021).
  17. Fischer, J., Heide, M., Huttner, W. B. Genetic modification of brain organoids. Frontiers in Cellular Neuroscience. 13, 558 (2019).
  18. Pașca, S. P., et al. A nomenclature consensus for nervous system organoids and assembloids. Nature. 609 (7929), 907-910 (2022).
  19. Kyrousi, C., Cappello, S. Using brain organoids to study human neurodevelopment, evolution and disease. Wiley Interdisciplinary Reviews: Developmental Biology. 9 (1), e347 (2020).
  20. Teriyapirom, I., Batista-Rocha, A. S., Koo, B. -. K. Genetic engineering in organoids. Journal of Molecular Medicine. 99 (4), 555-568 (2021).
  21. Saito, T., Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Developmental Biology. 240 (1), 237-246 (2001).
  22. Fukuchi-Shimogori, T., Grove, E. A. Neocortex patterning by the secreted signaling molecule FGF8. Science. 294 (5544), 1071-1074 (2001).
  23. Tabata, H., Nakajima, K. Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex. Neuroscience. 103 (4), 865-872 (2001).
  24. Kawasaki, H., Toda, T., Tanno, K. In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation. Biology Open. 2 (1), 95-100 (2012).
  25. Kawasaki, H., Iwai, L., Tanno, K. Rapid and efficient genetic manipulation of gyrencephalic carnivores using in utero electroporation. Molecular Brain. 5 (1), 24 (2012).
  26. Bai, J., et al. RNAi reveals doublecortin is required for radial migration in rat neocortex. Nature Neuroscience. 6 (12), 1277-1283 (2003).
  27. Kalebic, N., et al. CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo. EMBO reports. 17 (3), 338-348 (2016).
  28. Fischer, J., et al. Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids. EMBO Reports. 23 (11), e54728 (2022).
  29. Stauske, M., et al. Non-human primate iPSC generation, cultivation, and cardiac differentiation under chemically defined conditions. Cells. 9 (6), 1349 (2020).
  30. Mora-Bermúdez, F., et al. Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development. eLife. 5, e18683 (2016).
  31. Petkov, S., Dressel, R., Rodriguez-Polo, I., Behr, R. Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules. Cells. 9 (11), 2422 (2020).
  32. Camp, J. G., et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proceedings of the National Academy of Sciences of the United States of America. 112 (51), 15672-15677 (2015).
  33. Kanton, S., et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature. 574 (7778), 418-422 (2019).
  34. Lancaster, M. A., Knoblich, J. A. Generation of cerebral organoids from human pluripotent stem cells. Nature Protocols. 9 (10), 2329-2340 (2014).
  35. Cakir, B., et al. Engineering of human brain organoids with a functional vascular-like system. Nature Methods. 16 (11), 1169-1175 (2019).
  36. Masselink, W., et al. Broad applicability of a streamlined ethyl cinnamate-based clearing procedure. Development. 146 (3), dev166884 (2019).
  37. Denoth-Lippuner, A., Royall, L. N., Gonzalez-Bohorquez, D., Machado, D., Jessberger, S. Injection and electroporation of plasmid DNA into human cortical organoids. STAR Protocols. 3 (1), 101129 (2022).
  38. Denoth-Lippuner, A., et al. Visualization of individual cell division history in complex tissues using iCOUNT. Cell Stem Cell. 28 (11), 2020.e12-2034.e12 (2021).
  39. Kelava, I., Chiaradia, I., Pellegrini, L., Kalinka, A. T., Lancaster, M. A. Androgens increase excitatory neurogenic potential in human brain organoids. Nature. 602 (7895), 112-116 (2022).
  40. Lancaster, M. A., et al. Guided self-organization and cortical plate formation in human brain organoids. Nature Biotechnology. 35 (7), 659-666 (2017).
  41. Kalebic, N., et al. Neocortical expansion due to increased proliferation of basal progenitors is linked to changes in their morphology. Cell Stem Cell. 24 (4), 535.e9-550.e9 (2019).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved