We are grateful to the facilities of the CRTD and Dresden Concept partners for the outstanding support provided, notably K. Neumann and her team at the Stem Cell Engineering Facility, H. Hartmann and her team at the Light Microscopy Facility, A. Gompf and her team at the Flow Cytometry Facility and Hartmut Wolf of the MPI-CBG workshop for the construction of the electroporation chambers. We thank Joshua Schmidt for his feedback on the manuscript. MA acknowledges funding from the Center for Regenerative Therapies TU Dresden, the DFG (Emmy Noether, AL 2231/1-1), and the Schram Foundation.

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

The authors declare that they have no conflicts of interest.

Human cerebral and cortical organoids have become key models to investigate human neocortex development7,16,17. For modeling of human disorders, isogenic hiPSC lines and organoids have become the gold standard68. However, since the generation of novel iPSC lines is time-consuming and costly, the acute manipulation of cortical organoid models for studies of gene function has been widely applied.
<p cla...

The neocortex refers to the outer covering of the cerebral hemispheres and is a structure that is unique to mammals. The neocortex represents the seat of higher cognitive functions1,2,3,4,5. During development, neural stem and progenitor cells give rise to neurons in a process termed neurogenesis. Functional studies investigating human neocortex development provide the basis for elucidating the mechanisms underlying human neural stem cell regulation, neural pathologies, and human brain evolution2,6,7.
Historically, studies of human brain development relied on descriptive histological approaches using post-mortem tissue, with more recent free-floating tissue culture systems enabling functional investigations with human fetal tissue8,9. Additionally, human fetal brain tissue was shown to have the capacity to self-organize into long-term expanding organoids10. Fetal tissue research has provided important insights into human development11,12, yet restricted tissue availability and ethical considerations limit its widespread application for mechanistic studies of human brain development. In the past decade, protocols have been developed that allow the generation of three-dimensional neural organoids from human pluripotent stem cells (hPSC), including human induced PSC (hiPSC)13,14.
Cerebral organoids display important features of the developing brain, such as the formation of ventricle-like structures, apicobasal polarity, cortical cytoarchitecture, interkinetic nuclear migration during radial glia division, and neuronal migration. Importantly, these new human organoid models recapitulate characteristics of the human developing brain that are not modeled well in the mouse, including evolutionarily relevant key neural progenitor types, in particular basal radial glia (or outer radial glia)7,14,15. Several limitations of early cerebral organoid protocols - such as issues with organoid heterogeneity, limited nutrient supply to the inner core, and varied regional identity-have been addressed in recent protocol advancements and further improvements can be expected in the coming years15,16,17,18,19. Human cerebral and cortical organoids have quickly become key models to study human cortical development20, neurological disorders21,22,23,24 and brain evolution25,26,27,28,29, and to perform large-scale screening approaches30,31,32.
For acute genetic manipulation, two methods have primarily been used in animal models of neocortex development: viral delivery by infection of target cells33 and in utero or in vitro electroporation34,35. Injection of DNA - and, more recently, CRISPR/Cas9 ribonucleoprotein (RNP) complexes36- into the lateral ventricles, followed by electroporation, provides the advantage that specific regions of the brain can be targeted based on the orientation of the electroporation electrodes. Electroporation involves brief electric pulses that temporarily increase cell membrane permeability, allowing the introduction of DNA and other charged molecules into cells. In utero electroporation was first performed in the mouse37, where it rapidly became a widely applied methodology for developmental neurobiology. The method was subsequently also applied to other species, such as the rat38,39 and the ferret40,41,42, a gyrencephalic species used to study neocortex expansion and cortical folding3,43,44,45.
Electroporation has also become an important method in human brain organoid research46. In cerebral organoids, electroporation has been applied to visualize cell morphology and neuronal axons14,47, to deliver gene knockdown reagents22,48,49, and for investigation of gene function by overexpression50. The method is not restricted to human models but has also been applied for genetic modification of primate cerebral organoids50,51. Moreover, the electroporation of cortical organoids generated in a Spin&#160;&#937; spinning bioreactor has been described52.
In this protocol, we outline the electroporation of sliced human cortical organoids15 for studies of gene function in cortical development. Brain region-specific organoids increase reproducibility and consistency, which are critical for the success of quantitative analysis, for example, in disease modeling. In the sliced human cortical organoid protocol15, potent patterning cues are applied during iPSC differentiation to obtain a homogeneous population of dorsal forebrain progenitors, which is followed by the application of culture media that promote tissue growth with fewer instructive signals53,54. Slicing of cortical organoids has been shown to reduce cell death resulting from reduced availability of nutrients and oxygen in the organoid core15. Moreover, slicing supports the development of ventricle-like structures containing abundant basal radial glia, with sustained neurogenesis leading to the formation of an expanded cortical plate-like region15. The repeated slicing in this protocol also makes these cortical organoids particularly suitable to electroporation, as the ventricle lumens can be easily identified and targeted by injection. Electroporation of sliced cortical organoids has been applied to study gene regulatory regions and cortical evolution26,55.
During the electroporation procedure, the injection mix is delivered to the lumen of ventricle-like structures. Upon application of a pulsed electric field, the mix is taken up by apical radial glia that line the ventricle. As apical radial glia divide and give rise to more committed cell types4, the electroporated agents are passed on to basal progenitor cells and neurons. Electroporated progeny are distributed in the ventricular zone (VZ) and subventricular zone (SVZ) after 3 days and span most of the cortical wall, including the cortical plate (CP)-like region, at 7 days post-electroporation during mid-neurogenic stages26,55.
Here, we describe CRISPR/Cas9-mediated gene disruption56 by electroporation in sliced human cortical organoids26. In addition, the electroporation method can also be applied for gene overexpression, visualization of cell morphology and cellular processes by expression of fluorescent proteins, delivery of plasmid libraries for Massive Parallel Reporter Assays (MPRA), delivery of epigenome editing tools and labeling of cells for live imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL PP Centrifuge Tubes, conical</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>4D-Nucleofector Core Unit</td>
			<td>Lonza</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4D-Nucleofector X Unit</td>
			<td>Lonza</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polystyrene round-bottom tube with cell-strainer cap</td>
			<td>Corning</td>
			<td>Falcon 352235</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate, nunclon treated</td>
			<td>Thermo Fisher</td>
			<td>140675</td>
			<td>For hiPSC culture</td>
		</tr>
		<tr>
			<td>6-well plate, ultra low attachment</td>
			<td>Corning</td>
			<td>3471</td>
			<td>For organoid culture</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>STEMCELL Technologies</td>
			<td>72022</td>
			<td>Forebrain medium 1 (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Air compressor</td>
			<td>Aerotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Chicken IgY (IgG) (H+L)</td>
			<td>Jackson Immuno Research</td>
			<td>703-545-155</td>
			<td>Secondary antibody, RRID: AB_2340375; dilution 1:1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A-31572</td>
			<td>Secondary antibody, RRID: AB_162543; dilution 1:1000</td>
		</tr>
		<tr>
			<td>Alt-R CRISPR-Cas9 crRNA, 2 nmol</td>
			<td>Integrated DNA technologies</td>
			<td></td>
			<td>Custom design (order as 2 nmol)</td>
		</tr>
		<tr>
			<td>Alt-R CRISPR-Cas9 tracrRNA</td>
			<td>Integrated DNA technologies</td>
			<td>1072532</td>
			<td>5 nmol</td>
		</tr>
		<tr>
			<td>Alt-R S.p. HiFi Cas9 Nuclease V3</td>
			<td>Integrated DNA technologies</td>
			<td>1081060</td>
			<td>100 &#181;g, from Streptococcus pyogenes</td>
		</tr>
		<tr>
			<td>Amphotericin B (Fungizone)</td>
			<td>Gibco</td>
			<td>15290018</td>
			<td>Forebrain medium 2, 3, and 4&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Anti-GFP primary antibody (chicken, polyclonal)</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>RRID: AB_300798; dilution 1:2000</td>
		</tr>
		<tr>
			<td>Anti-PCGF4 primary antibody (mouse, monoclonal)</td>
			<td>Millipore</td>
			<td>05-637&#160;</td>
			<td>RRID: AB_309865; dilution 1:300</td>
		</tr>
		<tr>
			<td>ApoTome fluorescent microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Sigma Aldrich</td>
			<td>1043003</td>
			<td>Forebrain medium 4&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>B27-supplement (+ vitamin A)</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>Forebrain medium 3, 4&#160;&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Banana to Micrograbber Cable Kit</td>
			<td>Harvard Apparatus BTX</td>
			<td>45-0216</td>
			<td></td>
		</tr>
		<tr>
			<td>Biometra TRIO-Thermocycler (PCR machine)</td>
			<td>Analytik Jena</td>
			<td>846-2-070-723</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillaries, borosilicate glass with filament, OD 1.2 mm; ID 0.69 mm; 10 cm length</td>
			<td>Science Products</td>
			<td>BF120-69-10</td>
			<td>Need to be pulled to specific thickness</td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>STEMCELL Technologies</td>
			<td>72052</td>
			<td>Forebrain medium 2&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>CRTDi004-A</td>
			<td>Stem Cell Engineering Facility at CMCB DD</td>
			<td>https://hpscreg.eu/cell-line/CRTDi004-A</td>
			<td>Single-cell adapted hiPSC line from healthy donor</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td>1 mg/mL stock, use 1:1000 diluted for IF</td>
		</tr>
		<tr>
			<td>Dibutyryl-cAMP</td>
			<td>STEMCELL Technologies</td>
			<td>73884</td>
			<td>Forebrain medium 4&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES</td>
			<td>Fisher Scientific</td>
			<td>31330095</td>
			<td>Forebrain medium 1, 2, and 3&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>STEMCELL Technologies</td>
			<td>72102</td>
			<td>Forebrain medium 1&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Dumont #55 Forceps, straigth, 11 cm</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM 830 Square Wave Electroporation System</td>
			<td>Harvard Apparatus BTX</td>
			<td>45-2052</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma Aldrich</td>
			<td>32205-1L-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma Aldrich</td>
			<td>EDS-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma Aldrich</td>
			<td>F7252</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>GE Healthcare&#160;</td>
			<td>SH30070.03&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>For pulling of microcapillaries</td>
		</tr>
		<tr>
			<td>Geneious Prime</td>
			<td>GraphPad Software LLC d.b.a Geneious</td>
			<td>Software version 2024.0.5</td>
			<td></td>
		</tr>
		<tr>
			<td>gLACZ</td>
			<td>Kalebic et al., 2016; Platt et al., 2014</td>
			<td></td>
			<td>5&#39;-TGCGAATACGCCCACGCGATCGG; underlined nucleotides = PAM</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco&#160;</td>
			<td>35050038</td>
			<td>Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma Aldrich</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>gPCGF4 KO1</td>
			<td>this paper</td>
			<td></td>
			<td>5&#39;-TGAACTTGGACATCACAAATAGG (corresponds to the KO images in Figure 3I+J)</td>
		</tr>
		<tr>
			<td>gPCGF4 KO2</td>
			<td>this paper</td>
			<td></td>
			<td>5&#39;-ACAAATAGGACAATACTTGCTGG (corresponds to the KO images in Figure 3I+J)</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>made in house</td>
			<td></td>
			<td>1 M stock</td>
		</tr>
		<tr>
			<td>Horse Serum, heat-inactivated</td>
			<td>Gibco</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GDNF Recombinant Protein</td>
			<td>Thermo Fisher</td>
			<td>450-10</td>
			<td>Forebrain medium 4&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Human/Mouse/Rat BDNF Recombinant Protein</td>
			<td>Thermo Fisher</td>
			<td>450-02</td>
			<td>Forebrain medium 4&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma Aldrich</td>
			<td>258148</td>
			<td>To make TrisHCl</td>
		</tr>
		<tr>
			<td>ImmEdge (wax) Pen</td>
			<td>Vector Laboraories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin solution human</td>
			<td>Sigma Aldrich</td>
			<td>&#160;I9278</td>
			<td>Forebrain medium 3&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP2618-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacement</td>
			<td>Gibco&#160;</td>
			<td>10828-010</td>
			<td>Forebrain medium 1&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal 980 Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Melting Point Agarose, ultra pure</td>
			<td>Thermo Fisher</td>
			<td>16520100</td>
			<td>for embedding of hCOs during vibratome sectioning</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free</td>
			<td>Corning</td>
			<td>354230</td>
			<td>For organoid culture</td>
		</tr>
		<tr>
			<td>Matrigel hESC-Qualified Matrix, LDEV-free</td>
			<td>Corning</td>
			<td>354277</td>
			<td>For hiPSC culture</td>
		</tr>
		<tr>
			<td>MgCl2, 1 M</td>
			<td>Thermo Fisher</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector PicoPump + Foot Switch</td>
			<td>World Precision Instruments</td>
			<td>SYS-PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader Pipette Tips 0.5 to 20 &#181;L</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>For loading of glass capillaries</td>
		</tr>
		<tr>
			<td>Mowiol 4-88</td>
			<td>Sigma Aldrich</td>
			<td>81381</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR 1</td>
			<td>STEMCELL Technologies</td>
			<td>85850</td>
			<td>Stem cell medium</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>Forebrain medium 2, and 3&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB Next High Fidelity 2x PCR Mastermix&#160;</td>
			<td>New England Biolabs</td>
			<td>M0541S</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer counting chamber</td>
			<td>Brand</td>
			<td>718605</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Forebrain medium 4&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids</td>
			<td>Gibco&#160;</td>
			<td>11140050</td>
			<td>Forebrain medium 1, 2, 3, and 4 (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Nuclease-Free Duplex Buffer</td>
			<td>Integrated DNA technologies</td>
			<td>1072570</td>
			<td></td>
		</tr>
		<tr>
			<td>O. C. T. Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>Embedding medium for frozen tissue specimen</td>
		</tr>
		<tr>
			<td>P3 Primary Cell 4D-Nucleofector X Kit S</td>
			<td>Lonza</td>
			<td>V4XP-3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher Chemical</td>
			<td>P/0840/53</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GFP</td>
			<td>Addgene</td>
			<td>11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Peel-A-Way Embedding Mold TruncatedT12</td>
			<td>Polysciences Inc.</td>
			<td>18986-1</td>
			<td>For embedding of hCOs for vibratome and cryo-sectioning</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)&#160;</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Forebrain medium 1, 2, 3, and 4&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15 and for stem cell medium after nucleofection</td>
		</tr>
		<tr>
			<td>Petri Dish 60 mm x 15 mm, No Vent, Sterile</td>
			<td>Corning</td>
			<td>BP50-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish Platinum Electrode Chamber, 5 mm gap</td>
			<td>Harvard Apparatus BTX</td>
			<td>45-0504</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>made in house</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma Aldrich</td>
			<td>P2308</td>
			<td>From Titrachium album; 10 mg/mL stock</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28706</td>
			<td></td>
		</tr>
		<tr>
			<td>RHO/ROCK pathway inhibitor (Y-27632)</td>
			<td>STEMCELL Technologies</td>
			<td>72308</td>
			<td></td>
		</tr>
		<tr>
			<td>SB-431542</td>
			<td>STEMCELL Technologies</td>
			<td>72232</td>
			<td>Forebrain medium 2&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
		<tr>
			<td>Sodium Citrate Dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>W302600-1KG-K</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Sigma Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose&#160;</td>
			<td>Sigma Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus adhesion slides</td>
			<td>Fisher Scientific</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>SZX10 with a KL 300 LED</td>
			<td>Olympus</td>
			<td>SZX10</td>
			<td>Or alternative stereoscope</td>
		</tr>
		<tr>
			<td>Thermo Shaker</td>
			<td>Grant bio</td>
			<td>PSC24N</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma Aldrich</td>
			<td>T9284</td>
			<td>a.k.a. Octoxinol 9</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td>To make TrisHCl</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme (1x)</td>
			<td>Thermo Fisher</td>
			<td>12604021</td>
			<td>Dissociation enzmye to make single-cell suspensions</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td>a.k.a. Polysorbate 20</td>
		</tr>
		<tr>
			<td>Tyrode&#39;s salt</td>
			<td>Sigma Aldrich</td>
			<td>T2145-10x1l</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating Microtome (vibratome)</td>
			<td>Leica</td>
			<td>VT1200 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-Bore 1000 &#181;L Universal Fit Filter Tips</td>
			<td>Corning</td>
			<td>TF-1005-WB-R-S</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco&#160;</td>
			<td>21985-023</td>
			<td>Forebrain medium 1, 3, and 4&#160; (https://doi.org/10.1016/j.stem.2020.02.002)15</td>
		</tr>
	</tbody>
</table>

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.

CRISPR/Cas9-mediated gene ablation requires the design of gRNAs. Generally, targeting one of the first exons of a gene of interest with a pair of gRNAs spaced 7-11 bp apart (avoiding multiples of 3 bp) works well (Figure 1A). As a first test, the efficiency of template recognition by gRNAs may be interrogated in vitro using a PCR template26,40,62. Efficient targeting and Cas9-mediated cutti...

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

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. ...

electroporation-sliced-human-cortical-organoids-for-studies-gene

Research

JoVE Journal

Developmental Biology

855 Views.  TUD Dresden University of Technology. 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. 

Video: Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Genetic Modification of Primate Cerebral Organoids

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.

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.

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 ...

Human Ovarian Epithelium Organoids: A Model for Tissue Repair and Drug Testing

Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. 
Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.

Author Spotlight: Advancing Human Ovarian Repair and Cancer Studies with Surface Epithelium Organoids

This protocol describes establishing three-dimensional (3D) tissue organoids from primary human ovarian surface epithelium (hOSE) cells. The protocol includes isolation of hOSE from freshly collected ovaries, cellular expansion of the hOSE, cryopreservation-thawing procedures, and organoid derivation. Immunofluorescence, quantitative analysis, and showcasing utility as a screening platform are included.

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound ...

Methods Article

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function