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 R&#252;diger 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 C&#233;sar 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 supported by an ERC starting grant (101039421).

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,&#160;and common marmoset (cj_160419_5)31 iPSC lines. The culture conditions are summarized in Table 1. See the Table of Materials for details related to all the materials, reagents, and equipment used in this protocol.
<ol>
	<li>For culturing the respective iPSCs, follow the originally described culture conditions. In general, for the successful generation and electroporation of cerebral organoids, use iPSC lines that have not been cultured for more than 90 passages. Additionally, at the start of cerebral organoid generation, ensure that iPSCs exhibit features of pluripotency with no signs of differentiation.</li>
</ol>
2. Generation of cerebral organoids from primate iPSCs
NOTE: The protocol for cerebral organoid generation is based on a modified version28,30,32,33 of the original cerebral organoid protocol10,34 with some species-specific modifications (detailed below).
<ol>
	<li>Seeding of iPSCs to generate embryoid bodies (EBs)
	<ol>
		<li>Once the iPSCs have reached 80%-90% confluency, wash them with Dulbecco&#39;s phosphate-buffered saline (DPBS), and add 500 &#181;L of recombinant trypsin substitute or 1 mL of proteolytic and collagenolytic mixture. 
		NOTE: Typically, iPSCs are cultured in a 60 mm cell culture dish to obtain approximately 900,000 cells, which is enough to generate 96 cerebral organoids. The cell numbers can be adjusted depending on the number of organoids needed. Be aware that not all the generated cerebral organoids might be suitable for electroporation.</li>
		<li>Incubate the dish at 37 &#176;C for 2 min to detach the cells. 
		NOTE: Up to an additional 2 min of incubation at 37 &#176;C might be needed depending on the iPSC line or enzyme used. It is advised to examine the dish under a microscope to ensure that the cells have started detaching.</li>
		<li>Add 1.5 mL of prewarmed (37 &#176;C) iPSC culture medium to stop the reaction, and pipette up and down 7x-10x (not more than 10x) to dissociate the cells from the cell culture dish and to obtain a single-cell suspension.</li>
		<li>Transfer the cell suspension to a 15 mL conical centrifuge tube, and centrifuge the cells at 200 &#215; g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant, and resuspend the pellet in 2 mL of iPSC culture medium supplemented with either 50 &#181;M Y27632 or 50 &#181;M pro-survival compound.</li>
		<li>Use 10 &#181;L of the cell suspension to count the cells using a Neubauer chamber.</li>
		<li>Adjust the cell suspension to a concentration of 9,000 cells per 150 &#181;L (60,000 cells/mL) using iPSC culture medium supplemented with 50 &#181;M Y27632 or 50 &#181;M pro-survival compound.</li>
		<li>To generate embryoid bodies (EBs), seed 150 &#181;L of the cell suspension into each well of an ultra-low attachment 96-well plate. While pipetting, gently shake the tube containing the cell suspension to prevent the cells from sedimenting.</li>
		<li>Culture the EBs in a humified atmosphere of 5% CO2 and 95% air at 37 &#176;C (0 days post seeding [dps]). Do not disturb the EBs within the first 24 h after seeding. 
		NOTE: EBs generated from marmoset iPSCs need to be cultured in a humified atmosphere under hypoxic conditions (5% CO2, 5% O2, and 90% N2) at 37 &#176;C.</li>
		<li>After ~48 h (2 dps), change the medium to iPSC culture medium without Y27632/pro-survival compound. Remove 100 &#181;L of medium per well, and add 150 &#181;L of prewarmed (37 &#176;C) fresh medium without Y27632. Go row by row.</li>
		<li>Perform further medium changes every other day; remove 150 &#181;L of medium from each well, and add 150 &#181;L of prewarmed (37 &#176;C) fresh medium without Y27632/pro-survival compound per well. 
		NOTE: After 4-5 dps, the periphery of the EBs should become translucent.</li>
	</ol>
	</li>
	<li>Induction of neuroectoderm 
	NOTE: In general, good-quality EBs should have smooth contours and translucent borders at this stage. The time points of neural induction slightly differ between primate species and iPSC lines. In the case of the cell lines used here (see section 1 and Table of Materials), neural induction for marmoset EBs usually needs to be started at 4 dps, for rhesus macaque at 5 dps, and for human and chimpanzee EBs at 4-5 dps (Figure 1A), depending on the state of the EBs (see above).
	<ol>
		<li>Remove 150 &#181;L of medium from each well of the first row of the 96-well plate, and add 150 &#181;L of prewarmed (37 &#176;C) neural induction medium (see Table 2) per well in the same row.</li>
		<li>Continue changing the medium as described above row by row for the whole 96-well plate. Perform further neural induction medium (NIM) changes every other day by removing 150 &#181;L of NIM from each well and adding 150 &#181;L of prewarmed (37 &#176;C) fresh NIM. 
		NOTE: From this point on, the marmoset EBs should be cultured under the same conditions as the other primate EBs (humified atmosphere of 5% CO2 and 95% air at 37 &#176;C).</li>
	</ol>
	</li>
	<li>Embedding in basement membrane matrix 
	NOTE: Once the EBs have developed a pronounced, translucent, radially organized neuroepithelium on the surface, structural support needs to be provided for the development of ventricle-like structures. This is achieved by embedding the EBs into a basement membrane matrix. Due to differences in development rates, marmoset and rhesus macaque EBs are ready for embedding already at 7 dps, while human and chimpanzee EBs are usually embedded at 8-9 dps. For simplicity, basement membrane matrix refers only to Matrigel in this protocol. However, Geltrex can be used as a replacement.
	<ol>
		<li>In preparation for embedding, UV-sterilize scissors, forceps, a small rack for 0.2 mL tubes, and three to six squares of parafilm treated with 70% (vol/vol) ethanol under the laminar flow hood for 15 min. Let the basement membrane matrix thaw on ice for several hours (~1.5 mL of basement membrane matrix is usually enough for 96 EBs). 
		NOTE: Always keep the basement membrane matrix on ice.</li>
		<li>Create a 4 x 4 dimple grid on the parafilm. Place the parafilm grid on the 0.2 mL tube rack so that the paper-enveloped side is facing up, and gently press a gloved finger against each hole of the rack.</li>
		<li>Remove the paper, and cut the dimple grid out of the parafilm square using scissors to adjust its size to fit into a 60 mm cell culture dish. Place the dimpled parafilm back on the 0.2 mL tube rack to provide a basis for the basement membrane matrix droplet generation.</li>
		<li>Using a pipette with a cut 200 &#181;L pipette tip, carefully transfer the EBs one after another from the well of the culture dish to the parafilm dimples.</li>
		<li>After moving 16 EBs to the grid, take a new 200 &#181;L pipette tip, and remove the remaining medium from the dimples.</li>
		<li>Pipette one drop (~15 &#181;L) of basement membrane matrix onto each dimple containing one EB.</li>
		<li>Take a 10 &#181;L pipette tip and quickly move the EBs into the center of each droplet without disturbing the droplet borders.</li>
		<li>Place the dimpled parafilm with the basement membrane matrix drops in a 60 mm cell culture dish, and incubate for 15-30 min at 37 &#176;C to allow the matrix to polymerize.</li>
		<li>To detach the matrix-embedded EBs from the parafilm, add 5 mL of differentiation medium (DM) without vitamin A (see Table 2) to the dish, and turn the parafilm square upside-down using forceps so that the side with the EBs is facing the bottom of the dish.</li>
		<li>Carefully shake the dish to make the basement membrane matrix drops containing the EBs detach from the parafilm. If some of them are still attached, take an edge of the parafilm square using forceps, and rapidly roll it up toward the center of the dish multiple times.</li>
		<li>Culture the cerebral organoids on an orbital shaker at 55 rpm in a humified atmosphere of 5% CO2 and 95% air at 37 &#176;C. Keep them in DM without vitamin A with medium changes every other day. To induce the production of neurons, switch to DM with vitamin A (retinoic acid, RA) after 13 dps for marmoset and rhesus macaque cerebral organoids or 14-15 dps for human and chimpanzee cerebral organoids (Figure 1A). From this point on, change the medium every 3-4 days. 
		NOTE: To support neuronal survival, DM with vitamin A can be supplemented with 20 &#181;g/mL human neurotrophin 3 (NT3), 20 &#181;g/mL brain-derived neurotrophic factor (BDNF), and 1 &#181;L/mL basement membrane matrix from 40 dps on.</li>
	</ol>
	</li>
</ol>
3. Electroporation of primate cerebral organoids
NOTE: From a technical point of view, the electroporation of cerebral organoids can be conducted as soon as the ventricle-like structures are pronounced enough to be targeted by microinjection. The optimal electroporation time window depends on the biological question and on the cell population(s) of interest. For example, if apical progenitors (APs) are the main target, then cerebral organoids at around 30 dps are already suitable. If basal progenitors (BPs) or neurons are the main targets, older cerebral organoids of more than 50 dps should be used (see, for example, Fischer et al.28).
<ol>
	<li>Preparation of the electroporation setup 
	NOTE: The electroporation efficiency is strongly affected by the size and the concentration of the electroporated plasmid(s) (see the discussion section for details).
	<ol>
		<li>Prepare a sufficient amount of electroporation mix for the control and gene of interest (GOI), for example, 10 &#181;L of electroporation mix for each control and GOI to electroporate approximately 30 cerebral organoids per condition. 
		NOTE: For the composition of the electroporation mixes, see Table 3.</li>
		<li>Prewarm (non-sterile) Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) and DM with vitamin A to 37 &#176;C. Prepare a small spatula and fine and normal scissors, and spray the instruments with 70% (vol/vol) ethanol. 
		NOTE: The following steps can be performed either under sterile or non-sterile conditions as the DM contains antibiotics (see Table 2). In our experience, the absence of sterility has never caused any contamination.</li>
		<li>Prepare 35 mm cell culture dishes, and connect the Petri dish electrode chamber to the electroporator. 
		NOTE: Petri dish electrode chambers are commercially available. However, they can be easily produced in a cost-efficient way (see Supplemental File 1).</li>
		<li>Using microloader tips, fill microinjection needles with 8 &#181;L of each electroporation mix. Cut the tips of the needles using fine scissors before the first use to achieve a stable flow. However, remove only a small part of the tip, as a blunt and wide tip can severely damage the organoids. 
		NOTE: Microinjection needles are either commercially available as pre-pulled microinjection needles or can be pulled in the lab if a needle puller is available. Follow the needle puller manufacturer&#39;s instructions to generate microinjection needles with a long taper and a 10 &#181;m tip diameter.</li>
	</ol>
	</li>
	<li>Microinjection and electroporation
	<ol>
		<li>Under a microscope, choose five cerebral organoids with smooth borders and clearly visible ventricle-like structures. Move them to a 35 mm cell culture dish containing prewarmed (37 &#176;C) DMEM/F12 using a cut 1,000 &#181;L pipette tip. 
		NOTE: Choose cerebral organoids with pronounced and accessible ventricle-like structures (see Figure 1B).</li>
		<li>To inject a ventricle-like structure, carefully insert the needle through its wall, and infuse it with the electroporation mix until visibly filled. Do not apply excessive pressure on ventricle-like structures to avoid them bursting. Proceed with six to eight ventricle-like structures of each cerebral organoid in this manner. 
		NOTE: If the needle becomes clogged during the microinjection process, the tip needs to be slightly trimmed.</li>
		<li>Transfer one microinjected cerebral organoid to the Petri dish electrode chamber with a small amount of DMEM/F12. Arrange the organoid in a way that the surfaces of the microinjected ventricle-like structures face toward the electrode connected to the positive pole of the electroporator. 
		NOTE: Orienting the structures in this way ensures that the cells are transfected on the side of the ventricle-like structure that is not affected by an adjacent structure.</li>
		<li>Electroporate the cerebral organoids one by one using the following settings: 5 pulses of 80 V, a pulse duration of 50 ms, and an interval of 1 s. Move the electroporated organoids to a new 35 mm cell culture dish filled with prewarmed (37 &#176;C) DMEM/F12. 
		NOTE: The electroporation settings might depend on the available square wave electroporator. These settings are optimized for the referenced electroporation system. Increasing the voltage can lead to the displacement of the cells.</li>
		<li>Proceed in the same manner with the next five cerebral organoids using the second electroporation mix (for example, GOI). 
		NOTE: Repeat steps 3.2.1-3.2.5 until the desired number of electroporated cerebral organoids is reached.</li>
		<li>If the microinjection and electroporation were conducted under non-sterile conditions, transfer the electroporated organoids to a sterile 35 mm cell culture dish under a laminar flow hood while moving as little DMEM/F12 as possible to the new cell culture dish.</li>
	</ol>
	</li>
	<li>Further culture and fixation of cerebral organoids
	<ol>
		<li>Culture the electroporated organoids in DM with vitamin A on an orbital shaker at 55 rpm in a humified atmosphere of 5% CO2 and 95% air at 37 &#176;C.</li>
		<li>On the next day after electroporation, check the cerebral organoids for successful electroporation under a conventional inverted fluorescence microscope. 
		NOTE: Depending on the length of the further culture after electroporation, different cell populations within the cerebral organoid are affected (see also the representative results section).</li>
		<li>Proceed with downstream applications after culturing the electroporated cerebral organoids for an amount of time suitable for the biological question of interest. 
		NOTE: Electroporated cerebral organoids can be processed for different downstream applications (e.g., fixation for immunofluorescence staining or snap-frozen for RNA isolation and qRT-PCR). Here, we describe the fixation of electroporated cerebral organoids.</li>
		<li>Transfer the electroporated organoids to a 15 mL conical centrifuge tube using a cut 1,000 &#181;l pipette tip, and remove excess medium.</li>
		<li>Add a sufficient amount of 4% paraformaldehyde (PFA) in DPBS (pH 7.5), and incubate for 30 min at room temperature. 
		CAUTION: PFA is classified as a human carcinogen and may cause irreparable health damage. Additional precaution measures including nitrile gloves and goggles are strongly recommended.</li>
		<li>Aspirate the PFA, add 5 mL of DPBS, shake a little, and aspirate the DPBS. Repeat this 2x. Store the organoids in DPBS at 4 &#176;C until further use. 
		NOTE: The protocol can be paused here, as PFA-fixed cerebral organoids can be stored at 4 &#176;C for several months. PFA-fixed electroporated organoids can be analyzed by cryosectioning and immunofluorescence staining10,28 or by whole-mount staining and clearing35,36. For example images, see the representative results section (see Table 4 for antibody details).</li>
	</ol>
	</li>
</ol>

Genetic Modification of Primate Cerebral Organoids

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294 (5544), 1071-1074 (2001).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li><li>Kawasaki, H., Toda, T., Tanno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genetic+manipulation+of+cortical+progenitors+in+gyrencephalic+carnivores+using+in+utero+electroporation.">In vivo genetic manipulation of cortical progenitors in gyrencephalic carnivores using in utero electroporation.</a> Biology Open. 2 (1), 95-100 (2012).</li><li>Kawasaki, H., Iwai, L., Tanno, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+genetic+manipulation+of+gyrencephalic+carnivores+using+in+utero+electroporation.">Rapid and efficient genetic manipulation of gyrencephalic carnivores using in utero electroporation.</a> Molecular Brain. 5 (1), 24 (2012).</li><li>Bai, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+reveals+doublecortin+is+required+for+radial+migration+in+rat+neocortex.">RNAi reveals doublecortin is required for radial migration in rat neocortex.</a> Nature Neuroscience. 6 (12), 1277-1283 (2003).</li><li>Kalebic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-induced+disruption+of+gene+expression+in+mouse+embryonic+brain+and+single+neural+stem+cells+in+vivo.">CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.</a> EMBO reports. 17 (3), 338-348 (2016).</li><li>Fischer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-specific+ARHGAP11B+ensures+human-like+basal+progenitor+levels+in+hominid+cerebral+organoids.">Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids.</a> EMBO Reports. 23 (11), e54728 (2022).</li><li>Stauske, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-human+primate+iPSC+generation,+cultivation,+and+cardiac+differentiation+under+chemically+defined+conditions.">Non-human primate iPSC generation, cultivation, and cardiac differentiation under chemically defined conditions.</a> Cells. 9 (6), 1349 (2020).</li><li>Mora-Berm&#250;dez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+and+similarities+between+human+and+chimpanzee+neural+progenitors+during+cerebral+cortex+development.">Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development.</a> eLife. 5, e18683 (2016).</li><li>Petkov, S., Dressel, R., Rodriguez-Polo, I., Behr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+switch+from+neurogenesis+to+pluripotency+during+marmoset+monkey+somatic+cell+reprogramming+with+self-replicating+mRNAs+and+small+molecules.">Controlling the switch from neurogenesis to pluripotency during marmoset monkey somatic cell reprogramming with self-replicating mRNAs and small molecules.</a> Cells. 9 (11), 2422 (2020).</li><li>Camp, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+cerebral+organoids+recapitulate+gene+expression+programs+of+fetal+neocortex+development.">Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (51), 15672-15677 (2015).</li><li>Kanton, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+single-cell+genomic+atlas+uncovers+human-specific+features+of+brain+development.">Organoid single-cell genomic atlas uncovers human-specific features of brain development.</a> Nature. 574 (7778), 418-422 (2019).</li><li>Lancaster, M. A., Knoblich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+cerebral+organoids+from+human+pluripotent+stem+cells.">Generation of cerebral organoids from human pluripotent stem cells.</a> Nature Protocols. 9 (10), 2329-2340 (2014).</li><li>Cakir, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+human+brain+organoids+with+a+functional+vascular-like+system.">Engineering of human brain organoids with a functional vascular-like system.</a> Nature Methods. 16 (11), 1169-1175 (2019).</li><li>Masselink, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+applicability+of+a+streamlined+ethyl+cinnamate-based+clearing+procedure.">Broad applicability of a streamlined ethyl cinnamate-based clearing procedure.</a> Development. 146 (3), dev166884 (2019).</li><li>Denoth-Lippuner, A., Royall, L. N., Gonzalez-Bohorquez, D., Machado, D., Jessberger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+and+electroporation+of+plasmid+DNA+into+human+cortical+organoids.">Injection and electroporation of plasmid DNA into human cortical organoids.</a> STAR Protocols. 3 (1), 101129 (2022).</li><li>Denoth-Lippuner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+individual+cell+division+history+in+complex+tissues+using+iCOUNT.">Visualization of individual cell division history in complex tissues using iCOUNT.</a> Cell Stem Cell. 28 (11), 2020.e12-2034.e12 (2021).</li><li>Kelava, I., Chiaradia, I., Pellegrini, L., Kalinka, A. T., Lancaster, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgens+increase+excitatory+neurogenic+potential+in+human+brain+organoids.">Androgens increase excitatory neurogenic potential in human brain organoids.</a> Nature. 602 (7895), 112-116 (2022).</li><li>Lancaster, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+self-organization+and+cortical+plate+formation+in+human+brain+organoids.">Guided self-organization and cortical plate formation in human brain organoids.</a> Nature Biotechnology. 35 (7), 659-666 (2017).</li><li>Kalebic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortical+expansion+due+to+increased+proliferation+of+basal+progenitors+is+linked+to+changes+in+their+morphology.">Neocortical expansion due to increased proliferation of basal progenitors is linked to changes in their morphology.</a> Cell Stem Cell. 24 (4), 535.e9-550.e9 (2019).</li></ol>

The authors declare that they have no conflicts of interest.

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...

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.

<table><tbody><tr><td>20 &amp;micro;L Microloader</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Merck</td><td>8.05740.0005</td><td></td></tr><tr><td>35 mm cell culture dishes</td><td>Sarstedt</td><td>83.3900</td><td></td></tr><tr><td>60 mm cell culture dishes</td><td>CytoOne</td><td>CC7682-3359</td><td></td></tr><tr><td>Activin A</td><td>Sigma-Aldrich</td><td>SRP3003</td><td></td></tr><tr><td>AOC1</td><td>Selleckchem</td><td>S7217</td><td></td></tr><tr><td>Axio Observer.Z1 Inverted Fluorescence Microscope</td><td>Zeiss</td><td></td><td>replacable by comparable fluorescent microscopes</td></tr><tr><td>AZD0530</td><td>Selleckchem&amp;nbsp;</td><td>S1006</td><td></td></tr><tr><td>B-27 Supplement with Vitamin A (retinoic acid, RA) (50x)</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>B-27 Supplement without Vitamin A (50x)</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>BTX ECM 830 Square Wave Electroporation System</td><td>BTX</td><td>45-2052</td><td></td></tr><tr><td>CGP77675</td><td>Sigma-Aldrich</td><td>SML0314</td><td></td></tr><tr><td>Chimpanzee induced pluripotent stem cell line Sandra A</td><td></td><td></td><td>doi: 10.7554/elife.18683&amp;nbsp;</td></tr><tr><td>Common marmoset induced pluripotent stem cell line cj_160419_5</td><td></td><td></td><td>doi: 10.3390/cells9112422</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12)</td><td>Gibco</td><td>11320-033</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>14190-094</td><td>pH 7.0&amp;minus;7.3; warm to room temperature before use</td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7252-5G</td><td></td></tr><tr><td>Forskolin</td><td>Selleckchem</td><td>2449</td><td></td></tr><tr><td>GlutaMAX Supplement (100x)</td><td>Gibco</td><td>35050-061</td><td>glutamine substitute supplement</td></tr><tr><td>Heparin (1 mg/mL stock)</td><td>Sigma-Aldrich</td><td>H3149</td><td></td></tr><tr><td>Human induced pluripotent stem cell line iLonza2.2</td><td></td><td></td><td>doi: 10.3390/cells9061349</td></tr><tr><td>Human Neurotrophin-3 (NT-3)</td><td>PeproTech</td><td>450-03</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>19278</td><td></td></tr><tr><td>IWR1</td><td>Sigma-Aldrich</td><td>I0161</td><td></td></tr><tr><td>Leica MS5 stereomicroscope (MDG 17 transmitted-light base)</td><td>Leica</td><td>10473849</td><td>replacable by comparable stereomicroscopes</td></tr><tr><td>Matrigel</td><td>Corning</td><td>354277/354234</td><td>basement membrane matrix; alternatively, Geltrex (ThermoFisher Scientific, A1413302) can be used</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (100x)</td><td>Sigma-Aldrich</td><td>M7145</td><td></td></tr><tr><td>N-2 Supplement (100x)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich</td><td>P7793</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Merck</td><td>818715</td><td>handle with causion due to cancerogenecity</td></tr><tr><td>Penicillin/Streptomycin (10,000 U/mL)</td><td>PanBiotech</td><td>P06-07100</td><td></td></tr><tr><td>Petri dish electrode chamber</td><td>self-produced (see Supplemental File 1)</td><td></td><td>also commertially available</td></tr><tr><td>Pre-Pulled Glass Pipettes</td><td>WPI</td><td>TIP10LT</td><td>borosilicate glass pipettes with long taper, 10 &amp;micro;m tip diameter</td></tr><tr><td>Pro-Survival Compound</td><td>MerckMillipore</td><td>529659</td><td></td></tr><tr><td>Recombinant Human/Murine/RatBrain-Derived Neurotrophic Factor (BDNF)</td><td>PeproTech</td><td>AF-450-02</td><td></td></tr><tr><td>Rhesus macaque induced pluripotent stem cell line iRh33.1</td><td></td><td></td><td>doi: 10.3390/cells9061349</td></tr><tr><td>StemMACS iPS-Brew XF</td><td>Miltenyi Biotech</td><td>130-104-368</td><td></td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>Gibco</td><td>A1110501</td><td>proteolytic and collagenolytic enzyme mixture</td></tr><tr><td>TrypLE</td><td>Gibco</td><td>12604-013</td><td>recombinant trypsin substitute; warm to room temperature before use</td></tr><tr><td>Ultra-Low Attachment 96-well plates</td><td>Costar</td><td>7007</td><td></td></tr><tr><td>Y27632</td><td>Stemcell Technologies</td><td>72305</td><td></td></tr></tbody></table>

targeted microinjection and electroporation of primate cerebral organoids for genetic modification

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.

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...

Watch this Scientific Journal Video about Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification at JoVE.com

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

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.

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

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 ...

targeted-microinjection-electroporation-primate-cerebral-organoids

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Developmental Biology

3.4K Views.  German Primate Center, Leibniz Institute for Primate Research. Ons onderzoeksgebied is de ontwikkeling en evolutie van de hersenen van primaten. We proberen de factoren te begrijpen die ten grondslag liggen aan neocorticale expansie bij primaten. Om deze vraag op een praktische en ethisch verantwoorde manier aan te pakken, gebruiken we hersenorganoïden als ons onderzoeksmodel.Verschillen in hersenontwikkeling tussen moderne mensen, ons en neanderthalers zijn onlangs naar voren gekomen. Deze verschillen zijn te wijten aan een klein aantal aminozuu...

Video: Auteur in de schijnwerpers: Gerichte micro-injectie en elektroporatie van cerebrale organoïden van primaten voor genetische modificatie  

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Neuroscience

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

A Human Cerebral Organoid Model of Neural Cell Transplantation

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. 
We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.

Here, we describe a protocol for the transplantation and tracking of labeled neural cells into human cerebral organoids.

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For ...

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

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