The video demonstrates a protocol for electroporation and microinjection of cerebral organoids. It includes steps for preparing electroporation mixes, injecting solutions, electroporating organoids, and assessing GFP-positive cells. The process highlights the examination of cell lineage and distribution in organoids over time, indicating the development of progenitor and neuronal cells.

electroporation of primate cerebral organoids

Genetic Modification of Primate Cerebral Organoids

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. These limitations lead, for example, to the poor translatability of mouse models of human diseases to the human situation, as described for certain cases of microcephaly (e.g., Zhang et al.3). Recently, transgenic non-human primates, which are an evolutionarily, functionally, and morphologically closer model of human neocortex development, have come into focus4,5,6,7,8 as they overcome many limitations of cell culture- and rodent-based models. However, the use of non-human primates in research is not only highly expensive and time-consuming but also raises ethical concerns. More recently, the development of brain organoid technology9,10 has emerged as a promising alternative that solves many of the limitations of previous models11,12,13,14,15,16.
Brain organoids are three-dimensional (3D), multicellular structures that emulate the main features of the cytoarchitecture and cell-type composition of one or multiple brain regions for a defined developmental time window11,12,13,14,17. These 3D structures are generated either from induced pluripotent stem cells (iPSCs) or, if available for the species of interest, from embryonic stem cells (ESCs). In general, two types of brain organoids can be distinguished based on the methodology used: unguided and regionalized (guided) brain organoids18. In generating the latter type of organoids, small molecules or factors are provided that guide the differentiation of the pluripotent stem cells to organoids of a particular brain region (e.g., forebrain organoids)18. By contrast, in unguided organoids, the differentiation is not guided by the addition of small molecules but rather relies exclusively on the spontaneous differentiation of the iPSCs/ESCs. The resulting brain organoids consist of cell types representing different brain regions (e.g., cerebral organoids)18. Brain organoids combine many key features of brain development with relatively cost- and time-efficient generation from any species of interest for which iPSCs or ESCs are available11,12,13,14. This makes brain organoids an excellent model for many kinds of neurobiological studies, ranging from evolutionary and developmental questions to disease modeling and drug testing15,16. However, addressing such questions using brain organoids strongly depends on the availability of different methods for genetic modification.
One key aspect of studying neocortex (patho)physiological development and its evolution is the functional analysis of genes and gene variants. This is usually achieved by (ectopic) expression and/or by knock-down (KD) or knock-out (KO) of those genes. Such genetic modifications can be classified into stable and transient genetic modification, as well as into the modifications being temporally and spatially restricted or not restricted. Stable genetic modification is defined by the introduction of a genetic alteration into the host genome that is passed on to all subsequent cell generations. Depending on the time point of genetic modification, it can affect all the cells of an organoid or can be restricted to certain cell populations. Most frequently, stable genetic modification is achieved in brain organoids at the iPSC/ESC level by applying lentiviruses, transposon-like systems, and the CRISPR/Cas9 technology (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20). This has the advantage that all cells of the brain organoid carry the genetic modification and that it is not temporally or spatially restricted. However, the generation and characterization of these stable iPSC/ESC lines are very time-consuming, often taking several months until the first modified brain organoids can be analyzed (reviewed by e.g., Fischer et al.17, Kyrousi et al.19, or Teriyapirom et al.20).
In contrast, transient genetic modification is defined by the delivery of genetic cargo (e.g., a gene expression plasmid) that does not integrate into the host genome. While this modification can, in principle, be passed on to subsequent cell generations, the delivered genetic cargo will be progressively diluted with each cell division. Therefore, this type of genetic modification is usually temporally and spatially restricted. Transient genetic modification can be carried out in brain organoids by adeno-associated viruses or by electroporation (reviewed by, e.g., Fischer et al.17, Kyrousi et al.19, and Teriyapirom et al.20), with the latter being described in detail in this article. In contrast to stable genetic modification, this approach is very fast and cost-efficient. Indeed, electroporation can be performed within minutes, and, depending on the target cell population(s), electroporated organoids are ready for analysis within days (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19). However, gross morphological changes of the brain organoid, such as differences in size, cannot be detected using this method, as this type of genetic modification is temporally and spatially restricted. This restriction can also be an advantage, for example, in the case of studying individual cell populations within the organoid or the effects on brain organoids at specific developmental time points (reviewed by, e.g., Fischer et al.17 and Kyrousi et al.19).
A classical approach to study gene function during brain development and evolution is in utero electroporation. In utero electroporation is a well-known and useful technique for the delivery of gene expression constructs into rodent21,22,23 and ferret24,25 brains. First, a solution containing the expression construct(s) of interest is microinjected through the uterine wall into a certain ventricle of the embryonic brain, depending on the region to be targeted. In the second step, electric pulses are applied to transfect the cells directly lining the targeted ventricle. This approach is not only limited to ectopic expression or the overexpression of genes, as it can also be applied in KD or KO studies by microinjecting short hairpin (shRNA) or CRISPR/Cas9 (in the form of expression plasmids or ribonucleoproteins [RNPs]), respectively26,27. However, the in utero electroporation of mouse, rat, and ferret embryos has the same limitations as described above for these animal models.
Ideally, one would like to perform in utero electroporation directly in primates. While this is, in principle, technically possible, in utero electroporation is not conducted in primates due to ethical concerns, high animal maintenance costs, and small litter sizes. For certain primates, such as great apes (including humans), this is not possible at all. However, these primates have the greatest potential for the study of human (patho)physiological neocortex development and its evolution. One solution to this dilemma is to apply the electroporation technique to primate brain organoids28.
This paper presents a protocol for the electroporation of a subtype of primate brain organoids, primate cerebral organoids. This approach allows the fast and cost-efficient genetic modification of cell populations within the ventricle-like structures of the organoids. Specifically, we describe a unified protocol for the generation of primate cerebral organoids from human (Homo sapiens), chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and common marmoset (Callithrix jacchus) iPSCs. Moreover, we describe the microinjection and electroporation technique in detail and provide &#34;go&#34; and &#34;no-go&#34; criteria for performing primate cerebral organoid electroporation. This approach is an effective tool for studying (patho)physiological neocortex development and its evolution in a model especially close to the human situation.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

Our field of research is primate brain development and evolution. We are trying to understand the factors underlying neocortical expansion in primates. To address this question in a practical and ethically justifiable way, we use brain organoids as our research model.Differences in brain development between modern humans, us, and neanderthals have recently emerged. These differences are due to a small number of amino acid changes in proteins with key roles in brain development. Cerebral organoids have been a crucial model system in these studies.The study of primate development from an evolutionary point of view conducting gain and loss of function studies is important. However, apes including humans naturally cannot be used for such experiments, so genetic modification of organoids plays a vital role in this field. While working as a postdoc in my lab, Michael Heide demonstrated that the human specific gene ARHGAP11B was a key player in increasing brain size during human evolution.As a group leader at the German Primate Center now, Michael contributed crucial insight by comparing human versus chimpanzee cerebral organoids. Our protocol offers a targeted approach for genetic modifications by specific micro injecting ventricle-like structures instead of commercial electroporation cuvettes. This approach uses a cost efficient setup including square wave electroporator and Petri dish electrode chamber instead of expensive nuclear effector solutions.While studying function and brain development in primates by genetic modification is technically possible, it is methodologically demanding, expensive, and not allowed for great apes. The electroporation of primate cerebral organoids provides a fast and cost efficient approach to introduce genetic modifications in a model close to primate brain development. In the future, we would like to focus on the characterization of differentially expressed genes in the cortex of different primate species.We will study these genes by the electroporation of primate cerebral organoids and analyze their potential effects on cortical progenitor cells.

Watch this Scientific Journal Video about Electroporation of Primate Cerebral Organoids at JoVE.com

Electroporation of Primate Cerebral Organoids  

Begin by preparing a sufficient amount of electroporation mix for the control and gene of interest. Connect the Petri dish electrode chamber to the electroporator. Then using Microloader tips, fill microinjection needles with 8 microliters of each electroporation mix.Cut the tips of the needles before use to achieve a stable flow using fine scissors. Ensure to remove only a small part of the tip as a blunt and wide tip can severely damage the organoids. Under a microscope, choose five cerebral organoids with smooth borders and visible ventricle-like structures.Then move them to a 35-millimeter cell culture dish containing prewarmed DMEM/F-12 using a cut 1000 microliter pipette tip. Now, carefully insert the needle through the wall of a ventricle-like structure and infuse it with the electroporation mix until visibly filled. Do not apply excessive pressure on ventricle-like structures to avoid bursting.Transfer one microinjected cerebral organoid to the Petri dish electrode chamber with a small amount of DMEM/F-12. Arrange the organoid so that the surfaces of the microinjected ventricle-like structures face toward the electrode connected to the positive pole of the electroporator. Now, electroporate the cerebral organoids one by one using five pulses of 80 volts for 50 milliseconds each with an interval of one second.If the microinjection and electroporation were performed under non-sterile conditions, move the electroporated organoids under a laminar flow hood to a new 35-millimeter cell culture dish filled with prewarmed DMEM/F-12. Then culture the electroporated organoids in DM with vitamin A on an orbital shaker at 55 RPM in a humified atmosphere of 5%carbon dioxide and 95%air at 37 degrees Celsius. The next day, after electroporation, check the cerebral organoids for successful electroporation under a conventional inverted fluorescence microscope.Transfer the electroporated organoids to a 15-milliliter conical centrifuge tube using a cut 1000 microliter pipette tip and remove excess medium. Add a sufficient amount of 4%paraformaldehyde in DPBS and incubate for 30 minutes at room temperature. At the end of the incubation, aspirate the paraformaldehyde.Then add 5 milliliters of DPBS, shake gently, and aspirate the DPBS. Store the organoids in DPBS at 4 degrees Celsius until further use. Two days after electroporation, GFP-positive cells are almost exclusively localized in the ventricular zone and are positive for Pax6, indicating that these cells are apical progenitors or newborn basal progenitors.After 10 days, the GFP-positive cells are localized in the basal regions. These cells are also positive for Pax6 or NeuN, which is indicative of basal progenitors or neurons, respectively. 3-D reconstruction of the electroporated cerebral organoids to obtain an impression of the 3-D distribution of the GFP-positive cells is shown.

electroporation-of-primate-cerebral-organoids

Research

JoVE Journal

Developmental Biology

356 Görüntüleme.  German Primate Center, Leibniz Institute for Primate Research. Kontrol ve ilgili gen için yeterli miktarda elektroporasyon karışımı hazırlayarak başlayın. Petri çanak elektrot haznesini elektroporatöre bağlayın. Daha sonra Microloader uçlarını kullanarak, mikroenjeksiyon iğnelerini her elektroporasyon karışımından 8 mikrolitre ile doldurun.İnce makas kullanarak sabit bir akış elde etmek için kullanmadan önce iğnelerin uçlarını kesin. Künt ve geniş bir uç organoidlere ciddi şekilde zarar verebileceğinden, ucun sade...